Abstract:

Fatty acid-based, pre-cure-derived biomaterials, methods of making the
biomaterials, and methods of using them as drug delivery carriers are
described. The fatty acid-derived biomaterials can be utilized alone or
in combination with a medical device for the release and local delivery
of one or more therapeutic agents. Methods of forming and tailoring the
properties of said biomaterials and methods of using said biomaterials
for treating injury in a mammal are also provided.

Claims:

1. A coating for a medical device, comprising:a cross-linked fatty acid
oil, and a therapeutic agent, wherein the fatty acid was partially
cross-linked before association with the therapeutic agent.

2. The coating of claim 1, wherein the therapeutic agent is contained
within the coating in such a manner that the therapeutic agent has an
enhanced release profile.

8. A fatty-acid based, pre-cure-derived coating for a medical device
comprising cross-linked fatty acids and glycerides, wherein the fatty
acids and glycerides have disordered alkyl groups, which cause the
coating to be flexible and hydratable.

10. A fatty-acid based, pre-cure-derived coating for a medical device,
wherein the coating comprises lactone and ester cross links, as indicated
by an infrared absorption spectrum having peaks at approximately
1740-1850 cm-1, respectively.

11. A fatty-acid based, pre-cure-derived coating for a medical device,
comprising a cross-linked, fatty acid-derived biomaterial, wherein
approximately 60-90% of the biomaterial is constituted by fatty acids
with molecular weights below 500.

12. A fatty-acid based, pre-cure-derived biomaterial suitable for
achieving modulated healing in a tissue region in need thereof, wherein
the biomaterial is administered in an amount sufficient to achieve said
modulated healing, wherein the modulated healing comprises a modulation
of platelet or fibrin deposition in or near said tissue region.

13. The biomaterial of claim 12, wherein the tissue region is the
vasculature of a subject.

14. A fatty-acid based, pre-cure-derived biomaterial suitable for
achieving modulated healing at a site of vascular injury in need thereof,
wherein the composition is administered in an amount sufficient to
achieve said modulated healing, wherein the modulated healing comprises a
modulation of at least one metric of organized tissue repair.

15. The fatty-acid based, pre-cure-derived biomaterial of claim 14,
wherein the vascular healing is the inflammatory stage of vascular
healing.

16. The fatty-acid based, pre-cure-derived biomaterial of claim 14,
wherein the organized tissue repair comprises platelet or fibrin
deposition at the site of vascular injury.

17. The fatty-acid based, pre-cure-derived biomaterial of claim 14,
wherein the modulation of at least one metric of organized tissue repair
is a delay in the healing process at a site of vascular injury.

18. The fatty-acid based, pre-cure-derived biomaterials of claims 12 or
14, wherein the composition is administered to the region in need thereof
via a catheter, balloon, stent, surgical mesh, surgical dressing, or
graft.

19. The coating of claim 1, wherein the coating is configured to produce a
glyceride upon metabolization in-vivo.

46. A preparation for deriving a coating for a medical device, the
preparation comprising:a pre-cured cross-linked fatty acid oil, wherein
the coating contains ester and lactone cross-links, and wherein a portion
of the preparation comprises a pre-cured natural oil.

47. The preparation of claim 46, further comprising a therapeutic agent.

48. The preparation of claim 46, wherein the preparation has a viscosity
of about 1.0.times.10.sup.5 to about 1.0.times.10.sup.7 cps.

49. The preparation of claim 46, wherein the preparation is further
dissolved in an organic solvent.

50. A method for producing a fatty-acid based, pre-cure-derived coating
for a medical device, wherein the method comprises:curing an
oil-containing starting material according to a first curing condition to
form a second material;combining a therapeutic agent with the second
material to form a third material;and curing the third material such that
a coating is produced.

51. The method of claim 50, wherein the therapeutic agent is combined with
an oil-containing material or organic solvent before combining with the
second material.

52. The method of claim 50, wherein the curing temperature of the first
curing condition and/or total curing duration exceed the degradation
temperature of the therapeutic agent.

53. The method of claim 50, wherein the first curing condition results in
appreciable formation of esters and lactones in the oil such that
substantial cross linking of fatty acids occurs during the second curing
condition.

54. The method of claim 50, wherein the curing temperature and duration is
adjusted to tailor the release profile of the therapeutic agent.

55. The method of claim 50, wherein vitamin E is added to the second
material.

56. The method of claim 50, wherein the third material is combined with an
organic solvent, and applied to a medical device before curing to form a
conformal coating.

57. The method of claim 50, wherein the third material is sprayed on a
medical device before curing to form a coating.

59. The method of claim 50, wherein the medical device is a stent, a
catheter, a surgical mesh or a balloon.

60. The second material produced by the method of claim 50.

61. The second material of claim 60, wherein the second material has a
viscosity of about 1.0.times.10.sup.5 to about 1.0.times.10.sup.7 cps.

62. The non-polymeric coating produced by the method of claim 50.

63. The method of claim 50, wherein the therapeutic agent is an
anti-proliferative drug.

64. The method of claim 50, wherein the therapeutic agent is an
anti-inflammatory agent.

65. The method of claim 56, wherein: the first curing condition is
tailored such that the second material, when applied to a medical device,
provides a non-conformal coating on the medical device; and wherein the
second curing condition is tailored such that the third material, when
applied to a coating, provides a conformal coating.

66. The method of claim 50 wherein the curing time for the first curing
condition can be substantially increased in order to reduce the curing
time required for the second curing condition to obtain desired
mechanical properties of the final coating.

67. The method of claim 50 wherein the curing time for the first curing
condition can be substantially increased in order to reduce the curing
time required for the second curing condition to obtain desired
mechanical properties and preserve a thermally sensitive drug to the
final coating.

70. A fatty-acid based, pre-cure-derived coating for a medical device,
comprising: a non-polymeric, partially cross-linked fatty acid, and a
therapeutic agent, wherein the therapeutic agent is contained within the
coating in such a manner that the therapeutic agent has an enhanced
release profile.

71. A preparation for deriving a coating for a medical device, the
preparation comprising: a non-polymeric, partially cross-linked fatty
acid, and a therapeutic agent, wherein the coating contains ester and
lactone cross-links.

72. A fatty-acid based, pre-cure-derived coating for a medical device,
comprising: a cross-linked fatty acid oil, and a therapeutic agent;
wherein the coating is prepared by curing a natural oil-containing
starting material to induce cross-linking of a portion of the fatty
acids; adding a therapeutic agent to the partially-cross linked fatty
acid oil to form a therapeutic agent-oil composition; and curing the
therapeutic agent-oil composition to induce additional cross links in the
fatty acids, such that the coating is formed.

74. The coating of claim 72, wherein the therapeutic agent is combined
with vitamin E before combining with the partially-cross linked fatty
acid oil, such that the therapeutic agent has an enhanced release
profile.

75. A stand-alone film comprising a pre-cured fatty acid.

76. The stand-alone film of claim 75, wherein the stand-alone film
comprises approximately 5-50% C16 fatty acids.

77. The stand-alone film of claim 75, wherein the stand-alone film
comprises 5-25% C14 fatty acids.

78. The stand-alone film of claim 75, wherein the stand-alone film
comprises 5-40% C16 fatty acids.

79. The stand-alone film of claim 75, further comprising vitamin E.

80. The stand-alone film of claim 75, wherein the film is bioabsorbable.

81. The stand-alone film of claim 75, wherein the film maintains
anti-adhesive properties.

82. The stand-alone film of claim 75, further comprising a therapeutic
agent.

84. The stand-alone film of claim 75, wherein the therapeutic agent is
combined with the fatty acid compound prior to formation of the film,
resulting in the therapeutic agent being interspersed throughout the
film.

85. A stand-alone film, comprising:a cross-linked fatty acid oil, and a
therapeutic agent;wherein the stand-alone film is prepared by curing a
natural oil-containing starting material to induce cross-linking of a
portion of the fatty acids;adding a therapeutic agent to the
partially-cross linked fatty acid oil to form a therapeutic agent-oil
composition; andcuring the therapeutic agent-oil composition to induce
additional cross links in the fatty acids, such that the stand-alone film
is formed.

86. A fatty-acid based, pre-cure-derived biomaterial comprising a
partially cross-linked fatty acid and a therapeutic agent, wherein the
therapeutic agent comprises at least 40%, by weight, of the biomaterial
composition.

87. The fatty-acid based, pre-cure-derived biomaterial of claim 86,
wherein the therapeutic agent comprises at least 50%, by weight, of the
biomaterial composition.

[0002]Vascular interventions, such as vascular reperfusion procedures,
balloon angioplasty, and mechanical stent deployment, can often result in
vascular injury following mechanical dilation and luminal expansion of a
narrowed vessel. Often, subsequent to such intravascular procedures,
neointimal proliferation and vascular injury remodeling occurs along the
luminal surface of the injured blood vessel; more specifically,
remodeling occurs in the heart, as well as in vulnerable peripheral blood
vessels like the carotid artery, iliac artery, femoral and popliteal
arteries. No known mechanical suppression means has been found to prevent
or suppress such cellular proliferation from occurring immediately
following vascular injury from mechanical reperfusion procedures. Left
untreated, restenosis commonly occurs following a vascular intervention
within the treated vessel lumen within weeks of a vascular injury.
Restenosis, induced by localized mechanical injury, causes proliferation
of remodeled vascular lumen tissue, resulting in re-narrowing of the
vessel lumen, which can lead to thrombotic closure from turbulent blood
flow fibrin activation, platelet deposition and accelerated vascular flow
surface injury. Restenosis pre-disposes the patient to a thrombotic
occlusion and the stoppage of flow to other locations, resulting in
critical ischemic events, often with morbidity.

[0003]Restenosis initiated by mechanical induced vascular injury cellular
remodeling can be a gradual process. Multiple processes, including fibrin
activation, thrombin polymerization and platelet deposition, luminal
thrombosis, inflammation, calcineurin activation, growth factor and
cytokine release, cell proliferation, cell migration and extracellular
matrix synthesis each contribute to the restenotic process. While the
exact sequence of bio-mechanical mechanisms of restenosis is not
completely understood, several suspected biochemical pathways involved in
cell inflammation, growth factor stimulation and fibrin and platelet
deposition have been postulated. Cell derived growth factors such as
platelet derived growth factor, fibroblast growth factor, epidermal
growth factor, thrombin, etc., released from platelets, invading
macrophages and/or leukocytes, or directly from the smooth muscle cells,
provoke proliferative and migratory responses in medial smooth muscle
cells. These cells undergo a change from the contractile phenotype to a
synthetic phenotype. Proliferation/migration usually begins within one to
two days post-injury and peaks several days thereafter. In the normal
arterial wall, smooth muscle cells proliferate at a low rate,
approximately less than 0.1 percent per day.

[0004]However, daughter cells migrate to the intimal layer of arterial
smooth muscle and continue to proliferate and secrete significant amounts
of extracellular matrix proteins. Proliferation, migration and
extracellular matrix synthesis continue until the damaged endothelial
layer is repaired, at which time proliferation slows within the intima,
usually within seven to fourteen days post-injury. The newly formed
tissue is called neointima. The further vascular narrowing that occurs
over the next three to six months is due primarily to negative or
constrictive remodeling.

[0005]Simultaneous with local proliferation and migration, inflammatory
cells derived from the medial layer of the vessel wall continually invade
and proliferate at the site of vascular injury as part of the healing
process. Within three to seven days post-injury, substantial inflammatory
cell formation and migration have begun to accumulate along the vessel
wall to obscure and heal over the site of the vascular injury. In animal
models, employing either balloon injury or stent implantation,
inflammatory cells may persist at the site of vascular injury for at
least thirty days. Inflammatory cells may contribute to both the acute
and protracted chronic phases of restenosis and thrombosis.

[0006]Today, a preferred approach to the local delivery of a drug to the
site of vascular injury caused by an intravascular medical device, such
as a coronary stent, is to place a drug eluting coating on the device.
Clinically, medical devices coated with a drug eluting coating comprised
of either a permanent polymer or degradable polymer and an appropriate
therapeutic agent have shown angiographic evidence that vascular wall
proliferation following vascular injury and/or vascular reperfusion
procedures can be reduced, if not eliminated, for a certain period of
time subsequent to balloon angioplasty and/or mechanical stent
deployment. Local delivery of a single sirolimus or taxol compound via a
drug eluting medical device has been shown to be effective at minimizing
or preventing cellular proliferation and cellular remodeling when applied
immediately after vascular injury. Various analogs of these two
anti-proliferative compound examples have also been shown experimentally
and clinically to exhibit similar anti-proliferative activity with
similar drug eluting coatings. However, anti-proliferative compounds such
as sirolimus and taxol, together with a polymeric drug eluting coating
have also been shown clinically to exhibit a number of toxic side
effects, during and after principal drug release from the drug eluting
coating. These chronic and or protracted side effects place limits on the
amount of drug that can actually be delivered over a given period of
time, as well as challenge the compatibility of the polymer coatings used
to deliver a therapeutic agent locally to the site of the vascular injury
when applied directly to a site of inflammation and or cellular
remodeling. In addition, local overdosage of compounds like sirolimus and
taxol can prevent, limit or even stop cellular remodeling or
proliferation in and around the localized tissue area of the medical
device. For example, a lack of endothelial cell coverage during the
interruption of cell proliferation throughout the vascular injury healing
process exhibits a high potential for luminal thrombosis whereby fibrin
and a constant deposition of platelets blanket the exposed and non-healed
medical device and/or damaged vascular wall. Without uninterrupted
systemic support or administration of an anti-platelet medication like
clopidogrel combined with an anti-clotting agent, such as ASA, prior to
and following deployment of a drug eluting medical device, such devices
have been shown clinically to thrombose and occlude within days of
deployment. In addition, although these commercially available drug
eluting polymer coatings employed on medical devices are generally
characterized as being biocompatible, the lack of chemical hydrolysis,
degradation and absorption of these polymer-based chemistries into
smaller, easier to metabolize chemical components or products have now
been clinically demonstrated to initiate a protracted localized
inflammatory response at the site of the vascular injury, which may lead
to unexpected thrombotic occlusion within days of stopping anti-platelet
medication.

[0007]Wound healing or response to in-vivo injury (e.g., hernia repair)
follows the same general biological cascade as in vascular injury (see,
e.g., Y. C. Cheong et al. Human Reproduction Update. 2001; Vol. 7, No. 6,
pgs 556-566). This cascade includes inflammation of native tissue
followed by migration and proliferation of cells to mitigate the
inflammatory response, including platelets and macrophages, and a
subsequent healing phase which includes fibrin deposition and the
formation of fibrin matrix followed by tissue remodeling. In the case of
hernia repair, abnormal peritoneal healing can occur when there is the
expression of inflammatory cytokines from macrophages (e.g., α-TNF)
that can result in an inability of the fibrin matrix to be properly
broken down and can result in the formation of adhesions (Y. C. Cheong et
al., 2001). Abdominal adhesions formed after hernia repair can result in
pain, bowel strangulation, infertility and in some cases death (Y. C.
Cheong et al., 2001).

[0008]The sustained nature of the thrombotic and inflammatory response to
injury makes it desirable to provide a biomaterial that can reduce the
incidence of inflammatory and foreign body responses after implantation.
It would also be preferable to have a biomaterial that provides release
of one or more therapeutic agents over a period of time in order to
minimize such cell activated responses. Additionally, such a biomaterial
would also preferably be metabolized via a bioabsorption mechanism.

SUMMARY OF THE INVENTION

[0009]What is desired is a biomaterial (e.g., a coating or stand-alone
film) that can be utilized alone or as a drug delivery carrier that
prevents or diminishes chronic inflammation due to either the therapeutic
agent or components of the coating. Furthermore, it is desirable that the
biomaterial release and deliver therapeutic agents in a sustained and
controlled fashion to local tissue. The present invention is directed
toward various solutions that address this need.

[0010]What is also desired is a biomaterial (e.g., a coating or
stand-alone film) that can be bioabsorbed by cells and that can deliver a
drug without inducing chronic localized inflammation to tissues (e.g.,
the peritoneal or vascular tissue) that have been injured mechanically or
by reperfusion injury, whereby the biomaterial (e.g., coating or
stand-alone film) and the therapeutic agent are ingested and metabolized
by the cell, as it consumes the hydrolysis products of the biomaterial
with the drug.

[0011]In various aspects, the biomaterial is a coating for a medical
device, or a stand alone film. The biomaterial can be a fatty acid-based,
pre-cure-derived biomaterial. In various embodiments, the fatty
acid-based, pre-cure-derived biomaterial is non-polymeric. In certain
instances, as described herein, the source of the fatty acid is an oil,
e.g., a fish oil. In such an instance, the fatty acid-based,
pre-cure-derived biomaterial can also be referred to as an "oil-based,
pre-cure-derived biomaterial."

[0012]In a particular aspect, the invention provides a fatty acid-based,
pre-cure-derived biomaterial (e.g., coating or stand-alone film) that
contains a pre-cure component. As described herein, a "pre-cure"
component refers to fatty acids (e.g., from fish oil) that are partially
cured (using heat, UV, etc.) to induce an initial amount of fatty-acid
oxidation and crosslinking to form a viscous fatty acid-derived gel. The
pre-cure component can be dissolved in a solvent, and sprayed onto a
device, e.g., a medical device. Once the pre-cure component is associated
with a medical device, the device can be used for treatment in a subject,
or it can be further exposed to additional curing conditions, which may
result in a smooth, conformal coating referred to herein as a "fatty-acid
based, pre-cure-derived biomaterial (coating)." The pre-cure and/or the
fatty-acid based, pre-cure-derived biomaterial can be characterized as a
gel.

[0013]The pre-cured fatty acid component (also referred to herein as
"pre-cured fatty acid component," or simply "pre-cure;" when the source
of the fatty acid is an oil, such as fish oil, it can also be referred to
as "pre-cured oil") can be added to a therapeutic agent, wherein the
therapeutic agent is optionally combined with an oil, and the resulting
combination can be further cured, thereby further cross-linking the fatty
acids of the oil, to provide a fatty-acid based, pre-cure derived
biomaterial, meaning a portion of the fatty acid-based biomaterial was
pre-cured before formulation, and then exposed to further curing in the
presence of a therapeutic agent. In one embodiment, the fatty-acid based,
pre-cure derived biomaterial has tailored drug release properties. When
the resulting pre-cure-derived biomaterial is used as a coating for a
medical device or as a stand-alone film, it may also be referred to
herein as a "fatty-acid based, pre-cure-derived coating" or a "fatty-acid
based, pre-cure-derived stand-alone film."

[0014]The process of creating a pre-cure (e.g., of a fish oil) has the
advantage of creating an initial platform of oxidized fatty acid
cross-links that will be hydrolyzed by human tissue. In some embodiments,
portions of the pre-cure curing process can be done in the absence of the
therapeutic agent, enabling addition of the agent later in the process.
In such embodiments, the pre-cure process can be conducted at a
temperature and/or over a time period that would otherwise have lead to
degradation of a thermally/chemically sensitive therapeutic agent of
interest (e.g., a rapamycin or cyclosporine derivative), except that the
agent is not present for such portions of the pre-cure process. This
process results in a partially cross-linked composition, with reduced
oxidizable reactive sites (e.g., C═C bonds), that contains no
therapeutic agent. After the pre-cure is formed, the therapeutic agent is
then added, and optionally vitamin E is also added. The vitamin E
component has the advantage of protecting the drug and pre-cured oil from
further oxidation, but does not inhibit further cross-linking (e.g.,
esterification) of the fatty acid and/or glyceride components of the oil.

[0015]Accordingly, in various aspects, the present invention provides
methods for producing a hydrophobic, cross-linked, pre-cure-derived
biomaterial, wherein the pre-cure-derived biomaterial is utilized with
one or more therapeutic agents, wherein the therapeutic agents have a
controlled loading and are released in a sustained manner as the coating
is absorbed. In various embodiments, provided are methods of tailoring
the drug release profile of a pre-cure-derived biomaterial by control of
the curing conditions used to produce the pre-cure-derived biomaterial
(e.g., coating or stand-alone film) from an oil containing starting
material, the use of a free radical scavenger in an oil containing
starting material from which the pre-cure-derived biomaterial is formed,
or combinations thereof. In various embodiments, the methods of the
present invention tailor the drug release properties of a fatty-acid
based, pre-cure-derived biomaterial (e.g., coating or stand-alone film)
by controlling the degree of cross-linking of fatty acids. In various
embodiments, the methods of the present invention tailor the drug
delivery properties of a pre-cure-derived biomaterial (e.g., coating or
stand-alone film) by controlling the level of fatty acids, tocopherols,
lipid oxidation products, and soluble components in the fatty acid-based,
pre-cure-derived biomaterial.

[0016]In various aspects, the present invention may provide fatty
acid-derived biomaterials with a pre-cured oil component (e.g., coating
or stand-alone film) comprising one or more therapeutic agents with a
tailored release profile for one or more of the therapeutic agents. Such
a material is referred to herein as a "fatty acid-based, pre-cure-derived
biomaterial." In various embodiments, the tailored release profile
comprises a sustained release profile. In various embodiments, the
tailored release profile properties are controlled by the level of fatty
acids, tocopherols, lipid oxidation products, and soluble components in
the fatty acid-based, pre-cure-derived biomaterial. In various aspects of
the present invention, the fatty acid-based, pre-cure-derived biomaterial
contains fatty acids, many of which originate as triglycerides. It has
previously been demonstrated that triglyceride byproducts, such as
partially hydrolyzed triglycerides and fatty acid molecules can integrate
into cellular membranes and enhance the solubility of drugs into cellular
membranes (M. Cote, J. of Controlled Release. 2004, Vol. 97, pgs 269-281;
C. P. Burns et al., Cancer Research. 1979, Vol. 39, pgs 1726-1732; R.
Beck et al., Circ. Res. 1998, Vol 83, pgs 923-931; B. Henning et al.
Arterioscler. Thromb. Vasc. Biol. 1984, Vol 4, pgs 489-797). Whole
triglycerides are known not to enhance cellular uptake as well as a
partially hydrolyzed triglyceride, because it is difficult for whole
triglycerides to cross cell membranes due to their relatively larger
molecular size. Vitamin E compounds can also integrate into cellular
membranes resulting in decreased membrane fluidity and cellular uptake
(P. P. Constantinides. Pharmaceutical Research. 2006; Vol. 23, No. 2,
243-255).

[0017]In various aspects, the present invention may provide a
pre-cure-derived biomaterial (e.g., coating or stand-alone film)
containing fatty acids, glycerides, lipid oxidation products and
alpha-tocopherol in differing amounts and ratios to contribute to a fatty
acid-based, pre-cure-derived biomaterial in a manner that provides
control over the cellular uptake characteristics of the fatty acid-based,
pre-cure-derived biomaterial and any therapeutic agents mixed therein.

[0018]In various aspects, the present invention may provide coated medical
devices having a fatty acid-based, pre-cure-derived biomaterial drug
release coating comprising one or more layers of said pre-cure-derived
biomaterial, wherein at least one of the pre-cure-derived biomaterial
layers contains one or more therapeutic agents. The coating can be a
hydrophobic, cross-linked pre-cure-derived biomaterial (derived, e.g.,
from fish oil, making it an "oil-derived, pre-cure-derived biomaterial").
In various embodiments, the coating is non-polymeric. In various
embodiments, the drug release coating hydrolyzes in vivo, into
substantially non-inflammatory compounds. In various embodiments, the
pre-cure-derived biomaterial is coated onto a medical device that is
implantable in a patient to effect long term local delivery of the
therapeutic agent to the patient. In various embodiments the delivery is
at least partially characterized by the total and relative amounts of the
therapeutic agent released over time. In various embodiments, the
tailored delivery profile is controlled by the level of lipid oxidation,
vitamin E and/or soluble components in the fatty acid-based,
pre-cure-derived biomaterial. In various embodiments, the delivery
profile is a function of the solubility and lipophilicity of the coating
components and therapeutic agent in-vivo. The pre-cure-derived
biomaterial can be a stand-alone film, gel, suspension, or emulsion that
has the properties discussed above.

[0019]In various embodiments, the present invention may provide fatty-acid
based, pre-cure-derived coatings where the drug release profile of the
coating is tailored through the provision of two or more coatings and
selection of the location of the therapeutic agent. The drug location can
be altered, e.g., by coating a bare portion of a medical device with a
first starting material and creating a first cured coating, then coating
at least a portion of the first cured-coating with the drug-oil
formulation to create a second overlayer coating. It is to be understood
that the process of providing two layers can be extended to provide three
or more layers, wherein at least one of the layers comprises a fatty
acid-based, pre-cure-derived biomaterial. In addition, one or more of the
layers can be drug releasing, and the drug release profile of such layers
can be tailored using the methods described herein.

[0020]In accordance with various embodiments of the present invention, the
pre-cure-derived biomaterial (e.g., coating or stand-alone film) contains
lipids. The pre-cure-derived biomaterial can be formed from an oil, such
as fish oil, starting material. The pre-cure-derived biomaterial (e.g.,
coating or stand-alone film) can contain saturated, unsaturated, or
polyunsaturated fatty acids. When the fatty acid-based, pre-cure-derived
biomaterial is cross-linked, it can contain omega-3 fatty acids. The
fatty acid-based, pre-cure-derived biomaterial can also contain
alpha-tocopherol, or vitamin E derivatives, and/or a therapeutic agent.

[0021]The coatings of the present invention can be formulated to contain a
variety of other chemicals and entities in addition to a therapeutic
agent, including, but not limited to, one or more of: a pharmaceutically
acceptable carrier, an excipient, a surfactant, a binding agent, an
adjuvant agent, and/or a stabilizing agent (including preservatives,
buffers and antioxidants). In one embodiment, alpha-tocopherol TPGS may
be added to the coatings of the present invention.

[0022]In various aspects, the present invention may provide methods for
treating injury in a mammal, such as, e.g., a human. In various
embodiments, the injury is a vascular injury. In various embodiments, the
methods comprise locally administering one or more therapeutic agents in
a therapeutically effective amount by sustained release of the one or
more therapeutic agents from a coating comprising a fatty acid-based,
pre-cure-derived biomaterial.

[0023]The teachings herein demonstrate that the cured coatings and
stand-alone films that comprise a fatty acid-based, pre-cure-derived
biomaterial provide the ability to regulate the release profile of
drug-loaded fatty acid-based, pre-cure-derived biomaterials from the
films or from implantable devices. In various embodiments, the release
profile can be controlled through changes in oil chemistry by varying
fatty acid-based, pre-cure-derived biomaterial (e.g., coating or
stand-alone film) composition and cure times. The teachings demonstrate
that the release of therapeutic compounds from pre-cure-derived
biomaterials (e.g., coating or stand-alone film) can be modified based on
altering the oil curing conditions, the oil starting material, length of
curing, and amount of cross-linking. The teachings demonstrate that the
cross-linking and gelation of the fatty-acid based, pre-cure-derived oil
coatings and fatty-acid based, pre-cure-derived stand-alone films can be
directly dependent on the formation of hydroperoxides in the oil
component, which increases with increasing temperature and degree of
unsaturation of the oil. Dissolution experiments have shown that drug
release and coating degradation are more rapid for the cross-linked
coatings produced using lower temperature curing conditions (e.g., around
150° F.) than higher temperature curing conditions (e.g., around
200° F.).

[0024]In another aspect, the invention provides a fatty-acid based,
pre-cure-derived coating for a medical device, comprising a cross-linked
fish oil. The fish oil can optionally include a therapeutic agent. The
coating can be prepared according to the methods described herein, such
that, when the coating does include a therapeutic agent, the coating
releases the therapeutic agent at a desired release rate in vivo.

[0025]In another aspect, the invention provides a coating for a medical
device, comprising a fatty acid and a therapeutic agent, wherein the
fatty acid was partially cross-linked before association with the
therapeutic agent. That is, the fatty acid was partially cured to induce
an initial amount of fatty-acid cross-linking, and then combined with the
therapeutic agent. The resulting composition can then be exposed to an
additional curing procedure, e.g., after being applied to a medical
device, thereby further cross-linking the fatty acids to form a coating.
In one embodiment, the therapeutic agent is contained within the coating
in such a manner that the therapeutic agent has an enhanced release
profile. As used herein, the phrase "enhanced release profile" refers to
the release profile of a therapeutic agent by a pre-cure-derived
biomaterial that is prepared using the methods of the current invention.
That is, as discussed herein, by preparing a pre-cure, adding a
therapeutic agent to the pre-cure, and further curing the therapeutic
agent-pre-cure composition, a therapeutic cross-linked biomaterial is
created that will release the therapeutic agent in a manner different
from a preparation that was not prepared according to the methods of the
invention (i.e., a fatty acid-derived biomaterial that was not prepared
with the use of a pre-cure composition).

[0026]For example, by first preparing a pre-cure in the absence of a
therapeutic agent, the therapeutic agent is not exposed to process
conditions that would otherwise lead to its degradation. The therapeutic
agent can then be added to the pre-cure for further processing. Because
of this preservation of the therapeutic agent structure, there is less
degradation of the therapeutic agent during manufacturing of the coating.
Accordingly, there exists a higher amount of therapeutic agent in the
coating that can be released, especially compared to a coating that was
prepared without a pre-cure (i.e., compared to a coating in which the
uncured, fatty-acid containing material is first combined with a
therapeutic agent, and then cured). Thus, the therapeutic agent's release
profile is "enhanced."

[0027]In one embodiment, the coating of the invention further comprises a
pre-cured glyceride. The coating can comprise 5-25% C14 fatty acids
and/or 5-30% C16 fatty acids. The coating can be configured to
produce a glyceride upon metabolization in-vivo. The coating can comprise
approximately 30-90% saturated fatty acids; approximately 30-80%
unsaturated fatty acids; a glyceride; one or more of the group consisting
of a glyceride, a glycerol, and a fatty alcohol, any of which can be
partially cross-linked; and/or vitamin E.

[0028]In another embodiment, the coating is associated with an implantable
device. The coating can be associated with a medical device, and the
medical device is a stent, a catheter, a surgical mesh, or a balloon.

[0029]In one embodiment, the therapeutic agent that is associated with the
coating is an anti-proliferative drug, an anti-inflammatory agent, an
antimicrobial agent or antibiotic agent. In another embodiment, the
therapeutic agent is Compound A, Compound B, Compound C, Compound D,
Compound E (as described below), a cyclosporine derivative or rapamycin
derivative.

##STR00001## ##STR00002##

[0030]In another embodiment, the coating has a release profile of the
therapeutic agent in 0.01 M phosphate buffered saline (PBS) out to about
5-20 days. The coating can release said therapeutic agent at a desired
release rate in vivo. In one embodiment, the coating has a release
profile of the therapeutic agent in 0.01 M phosphate buffered saline
(PBS) out to more than 20 days.

[0031]In another embodiment, the coating comprises approximately 10-20%
C14 saturated fatty acids and approximately 25-50% C16
saturated fatty acids. The coating can comprise lactone and ester cross
links. The coating can contain disordered hydrocarbon chains as
determined by infrared absorption and X-ray diffraction.

[0032]In another embodiment, the coating does not contain a cross-linking
agent.

[0033]In still another embodiment, the coating hydrolyzes in vivo into
fatty acids, glycerols, and glycerides; hydrolyzes in vivo into
non-inflammatory components; and/or contains an amount of carboxylic acid
groups sufficient to facilitate hydrolysis in vivo.

[0034]The coating can comprises approximately 50-90% saturated fatty
acids; approximately 10-50% unsaturated fatty acids; a glyceride; and/or
one or more of the group consisting of a glyceride, a glycerol, and a
fatty alcohol, any of which can be partially cross-linked. In one
embodiment, the source of the fatty acids is an oil, such as oil is a
fish oil, olive oil, grape oil, palm oil, or flaxseed oil. In one
embodiment, the source is a fish oil.

[0037]In another aspect, the invention provides a fatty-acid based,
pre-cure-derived coating for a medical device comprising cross-linked
fatty acids and glycerides, wherein the fatty acids and glycerides have
disordered alkyl groups, which cause the coating to be flexible and
hydratable.

[0039]In still another aspect, the invention provides a fatty-acid based,
pre-cure-derived coating for a medical device, wherein the coating
comprises lactone and ester cross links, as indicated by an infrared
absorption spectrum having peaks at approximately 1740-1850 cm-1,
respectively.

[0040]In another aspect, the invention provides a fatty-acid based,
pre-cure-derived coating for a medical device, comprising a cross-linked,
fatty acid-derived biomaterial, wherein approximately 60-90% of the
biomaterial is constituted by fatty acids with molecular weights below
500.

[0041]In yet another aspect, the invention provides a fatty-acid based,
pre-cure-derived biomaterial suitable for achieving modulated healing in
a tissue region in need thereof, wherein the biomaterial is administered
in an amount sufficient to achieve said modulated healing, wherein the
modulated healing comprises a modulation of platelet or fibrin deposition
in or near said tissue region. In one embodiment, the tissue region is
the vasculature of a subject.

[0042]In still another aspect, the invention provides a fatty-acid based,
pre-cure-derived biomaterial suitable for achieving modulated healing at
a site of vascular injury in need thereof, wherein the composition is
administered in an amount sufficient to achieve said modulated healing,
wherein the modulated healing comprises a modulation of at least one
metric of organized tissue repair. In one embodiment, the vascular
healing is the inflammatory stage of vascular healing. In another
embodiment, the organized tissue repair comprises platelet or fibrin
deposition at the site of vascular injury. In another embodiment, the
modulation of at least one metric of organized tissue repair is a delay
in the healing process at a site of vascular injury.

[0043]In another embodiment, the biomaterials of the invention are
administered to the region in need thereof via a catheter, balloon,
stent, surgical mesh, surgical dressing, or graft.

[0044]In another aspect, provided herein is a preparation for deriving a
coating for a medical device, the preparation comprising:

[0045]a pre-cured cross-linked fatty acid oil, wherein the coating
contains ester and lactone cross-links, and wherein a portion of the
preparation comprises a pre-cured natural oil. The preparation can
further comprise a therapeutic agent. The preparation has a viscosity of
about 1.0×105 to about 1.0×107 cps. The preparation
can be further dissolved in an organic solvent.

[0046]Also provided herein is a method for producing a fatty-acid based,
pre-cure-derived coating for a medical device, wherein the method
comprises:

[0047]curing an oil-containing starting material according to a first
curing condition to form a second material;

[0048]combining a therapeutic agent with the second material to form a
third material;

[0049]and curing the third material such that a coating is produced.

[0050]In one embodiment of the method, the therapeutic agent is combined
with an oil-containing material or organic solvent before combining with
the second material. In another embodiment, the curing temperature of the
first curing condition and/or total curing duration exceed the
degradation temperature of the therapeutic agent. In still another
embodiment, the first curing condition results in appreciable formation
of esters and lactones in the oil such that substantial cross linking of
fatty acids occurs during the second curing condition. In still another
embodiment, the curing temperature and duration is adjusted to tailor the
release profile of the therapeutic agent. Vitamin E can be added to the
second material. In another embodiment, the third material is combined
with an organic solvent, and applied to a medical device before curing to
form a conformal coating. The third material can then be sprayed on a
medical device before curing to form a coating, e.g., a non-conformal
coating. In another embodiment of the method, the oil-containing starting
material is fish oil. In still another embodiment of the method, the
medical device is a stent, a catheter, a surgical mesh or a balloon.

[0051]The second material produced by the method can have a viscosity of
about 1.0×105 to about 1.0×107 cps.

[0052]The therapeutic agent used in the method can be an
anti-proliferative drug or an anti-inflammatory agent. The therapeutic
agent used in the method can also be Compound A, Compound B, Compound C,
Compound D, Compound E, a cyclosporine derivative or rapamycin
derivative.

[0053]In another embodiment of the method, the first curing condition is
tailored such that the second material, when applied to a medical device,
provides a non-conformal coating on the medical device; and wherein the
second curing condition is tailored such that the third material, when
applied to a coating, provides a conformal coating. In another
embodiment, the curing time for the first curing condition can be
substantially increased in order to reduce the curing time required for
the second curing condition to obtain desired mechanical properties of
the final coating. In another embodiment, the first curing condition can
be substantially increased in order to reduce the curing time required
for the second curing condition to obtain desired mechanical properties
and preserve a thermally sensitive drug to the final coating.

[0054]In another aspect, provided herein is a fatty-acid based,
pre-cure-derived coating for a medical device, wherein said coating
comprises: a hydrophobic, non-polymeric cross-linked fish oil; and a
therapeutic agent; wherein the coating can withstand 16-22 psi of
compressive force.

[0055]In still another aspect, provided herein is a fatty-acid based,
pre-cure-derived medical device coating, that hydrolyzes in vivo into
fatty acids, glycerols, and glycerides.

[0056]In yet another aspect, provided herein is a fatty-acid based,
pre-cure-derived coating for a medical device, comprising: a
non-polymeric, partially cross-linked fatty acid, and a therapeutic
agent, wherein the therapeutic agent is contained within the coating in
such a manner that the therapeutic agent has an enhanced release profile.

[0057]In another aspect, provided herein is a preparation for deriving a
coating for a medical device, the preparation comprising: a
non-polymeric, partially cross-linked fatty acid, and a therapeutic
agent, wherein the coating contains ester and lactone cross-links.

[0058]In still another aspect, provided herein is a fatty-acid based,
pre-cure-derived coating for a medical device, comprising: a cross-linked
fatty acid oil, and a therapeutic agent; wherein the coating is prepared
by curing a natural oil-containing starting material to induce
cross-linking of a portion of the fatty acids; adding a therapeutic agent
to the partially-cross linked fatty acid oil to form a therapeutic
agent-oil composition; and curing the therapeutic agent-oil composition
to induce additional cross links in the fatty acids, such that the
coating is formed. In one embodiment of the coating, the therapeutic
agent is combined with a natural oil-containing material, organic solvent
and/or vitamin E before combining with the partially-cross linked fatty
acid oil. In another embodiment, the therapeutic agent is combined with
vitamin E before combining with the partially-cross linked fatty acid
oil, such that the therapeutic agent has an enhanced release profile.

[0059]In another aspect, provided herein is a stand-alone film comprising
a pre-cured fatty acid. The stand-alone film can comprise approximately
5-50% C16 fatty acids; 5-25% C14 fatty acids, 5-40% C16
fatty acids; and/or vitamin E. The film can be bioabsorbable, and/or
maintain anti-adhesive properties. The stand-alone film can further
comprise a therapeutic agent, such as Compound A, Compound B, Compound C,
Compound D, Compound E, a cyclosporine derivative or rapamycin
derivative. The therapeutic agent can be combined with the fatty acid
compound prior to formation of the film, resulting in the therapeutic
agent being interspersed throughout the film.

[0060]In another aspect, provided herein is a stand-alone film,
comprising:

[0061]a cross-linked fatty acid oil, and a therapeutic agent;

[0062]wherein the stand-alone film is prepared by curing a natural
oil-containing starting material to induce cross-linking of a portion of
the fatty acids;

[0063]adding a therapeutic agent to the partially-cross linked fatty acid
oil to form a therapeutic agent-oil composition; and

[0064]curing the therapeutic agent-oil composition to induce additional
cross links in the fatty acids, such that the stand-alone film is formed.

[0065]In another aspect, provided herein is a fatty-acid based,
pre-cure-derived biomaterial comprising a partially cross-linked fatty
acid and a therapeutic agent, wherein the therapeutic agent comprises at
least 40%, by weight, of the biomaterial composition. In another
embodiment, the therapeutic agent comprises at least 50%, by weight, of
the biomaterial composition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0066]The foregoing and other aspects, embodiments, objects, features and
advantages of the invention can be more fully understood from the
following description in conjunction with the accompanying drawings. In
the drawings, like reference characters generally refer to like features
and structural elements throughout the various figures. The drawings are
not necessarily to scale, emphasis instead being placed upon illustrating
the principles of the invention.

[0067]FIG. 1 is a schematic illustration of an example of the creation of
peroxide and ether cross-linking in a polyunsaturated oil;

[0068]FIG. 2 is a schematic illustration of an example of the creation
carbon-carbon cross-linking in a polyunsaturated oil (Diels-Alder type
reaction);

[0069]FIG. 3 shows the mechanism for the formation of the hydrophobic
pre-cured-derived biomaterial coating;

[0090]FIGS. 24A, 24B and 24C shows FTIR spectra of vitamin E dissolved in
MTBE and sprayed onto coupons with and without final curing;

[0091]FIGS. 25A, 25B and 25C show HPLC chromatograms of a vitamin E
control overlaid with vitamin E sprayed onto coupons before and after
curing;

[0092]FIGS. 26A, 26B and 26C show FTIR analysis of a therapeutic agent
after spraying onto coupons before and after curing;

[0093]FIGS. 27A, 27B and 27C show HPLC chromatograms of a therapeutic
agent after spraying onto coupons before and after curing;

[0094]FIGS. 28A, 28B and 28C presents the FTIR spectra of the Compound B
fatty acid-derived, pre-cured biomaterial coating before and after final
curing;

[0095]FIGS. 29A and 29B show HPLC chromatograms of Compound B control
overlaid with the Compound B assay results obtained from a pre-cure
derived biomaterial after final curing;

[0096]FIG. 30 shows fatty acid profile data acquired for a therapeutic
agent/biomaterial formulation sprayed on coupons with and without final
curing;

[0097]FIGS. 31A, 31B and 31C show FTIR spectra of a therapeutic agent in
75:25 pre-cured fish oil:vitamin E sprayed onto stents and cured at
various times; and

[0098]FIGS. 32, 33 and 34 show results from the in vivo experiments
described in Example 15.

DETAILED DESCRIPTION

[0099]The present invention is directed toward the formation of a
fatty-acid based, pre-cure-derived biomaterial that can be utilized alone
or in combination with a medical device for the release and local
delivery of one or more therapeutic agents, methods of forming and
tailoring the properties of said coatings and methods of using said
coatings for treating injury in a mammal. Additionally, due to the unique
properties of the underlying chemistry of the biomaterial, it will be
demonstrated that the coating contains specific chemical components that
assist in reducing a foreign body response and inflammation at the site
of tissue injury during implantation that improves its in-vivo
performance. The fatty-acid based, pre-cure-derived biomaterial can be
formed from a pre-cure, e.g., a pre-cured fatty acid.

[0100]Prior to further describing the invention, it may be helpful to
generally and briefly describe injury and the biological response
thereto.

Vascular Injury

[0101]Vascular injury causing intimal thickening can be broadly
categorized as being either biologically or mechanically induced.
Biologically mediated vascular injury includes, but is not limited to,
injury attributed to infectious disorders including endotoxins and herpes
viruses, such as cytomegalovirus; metabolic disorders, such as
atherosclerosis; and vascular injury resulting from hypothermia, and
irradiation. Mechanically mediated vascular injury includes, but is not
limited to, vascular injury caused by catheterization procedures or
vascular scraping procedures, such as percutaneous transluminal coronary
angioplasty; vascular surgery; transplantation surgery; laser treatment;
and other invasive procedures which disrupt the integrity of the vascular
intima or endothelium. Generally, neointima formation is a healing
response to a vascular injury.

Inflammatory Response

[0102]Wound healing upon vascular injury occurs in several stages. The
first stage is the inflammatory phase. The inflammatory phase is
characterized by hemostasis and inflammation. Collagen exposed during
wound formation activates the clotting cascade (both the intrinsic and
extrinsic pathways), initiating the inflammatory phase. After injury to
tissue occurs, the cell membranes, damaged from the wound formation,
release thromboxane A2 and prostaglandin 2-alpha, which are potent
vasoconstrictors. This initial response helps to limit hemorrhage. After
a short period, capillary vasodilatation occurs secondary to local
histamine release, and the cells of inflammation are able to migrate to
the wound bed. The timeline for cell migration in a normal wound healing
process is predictable. Platelets, the first response cell, release
multiple chemokines, including epidermal growth factor (EGF),
fibronectin, fibrinogen, histamine, platelet-derived growth factor
(PDGF), serotonin, and von Willebrand factor. These factors help
stabilize the wound through clot formation. These mediators act to
control bleeding and limit the extent of injury. Platelet degranulation
also activates the complement cascade, specifically C5a, which is a
potent chemoattractant for neutrophils.

[0103]As the inflammatory phase continues, more immune response cells
migrate to the wound. The second response cell to migrate to the wound,
the neutrophil, is responsible for debris scavenging, complement-mediated
opsonization of bacteria, and bacteria destruction via oxidative burst
mechanisms (i.e., superoxide and hydrogen peroxide formation). The
neutrophils kill bacteria and decontaminate the wound from foreign
debris.

[0104]The next cells present in the wound are the leukocytes and the
macrophages (monocytes). The macrophage, referred to as the orchestrator,
is essential for wound healing. Numerous enzymes and cytokines are
secreted by the macrophage. These include collagenases, which debride the
wound; interleukins and tumor necrosis factor (TNF), which stimulate
fibroblasts (produce collagen) and promote angiogenesis; and transforming
growth factor (TGF), which stimulates keratinocytes. This step marks the
transition into the process of tissue reconstruction, i.e., the
proliferative phase.

Cell Proliferation

[0105]The second stage of wound healing is the proliferative phase.
Epithelialization, angiogenesis, granulation tissue formation, and
collagen deposition are the principal steps in this anabolic portion of
wound healing. Epithelialization occurs early in wound repair. At the
edges of wounds, the epidermis immediately begins thickening. Marginal
basal cells begin to migrate across the wound along fibrin strands
stopping when they contact each other (contact inhibition). Within the
first 48 hours after injury, the entire wound is epithelialized. Layering
of epithelialization is re-established. The depths of the wound at this
point contain inflammatory cells and fibrin strands. Aging effects are
important in wound healing as many, if not most, problem wounds occur in
an older population. For example, cells from older patients are less
likely to proliferate and have shorter life spans and cells from older
patients are less responsive to cytokines.

[0106]Heart disease can be caused by a partial vascular occlusion of the
blood vessels that supply the heart, which is preceded by intimal smooth
muscle cell hyperplasia. The underlying cause of the intimal smooth
muscle cell hyperplasia is vascular smooth muscle injury and disruption
of the integrity of the endothelial lining. Intimal thickening following
arterial injury can be divided into three sequential steps: 1) initiation
of smooth muscle cell proliferation following vascular injury, 2) smooth
muscle cell migration to the intima, and 3) further proliferation of
smooth muscle cells in the intima with deposition of matrix.
Investigations of the pathogenesis of intimal thickening have shown that,
following arterial injury, platelets, endothelial cells, macrophages and
smooth muscle cells release paracrine and autocrine growth factors (such
as platelet derived growth factor, epidermal growth factor, insulin-like
growth factor, and transforming growth factor) and cytokines that result
in the smooth muscle cell proliferation and migration. T-cells and
macrophages also migrate into the neointima. This cascade of events is
not limited to arterial injury, but also occurs following injury to veins
and arterioles.

Granulomatous Inflammation

[0107]Chronic inflammation, or granulomatous inflammation, can cause
further complications during the healing of vascular injury. Granulomas
are aggregates of particular types of chronic inflamatory cells which
form nodules in the millimeter size range. Granulomas may be confluent,
forming larger areas. Essential components of a granuloma are collections
of modified macrophages, termed epithelioid cells, usually with a
surrounding zone of lymphocytes. Epithelioid cells are so named by
tradition because of their histological resemblance to epithelial cells,
but are not in fact epithelial; they are derived from blood monocytes,
like all macrophages. Epithelioid cells are less phagocytic than other
macrophages and appear to be modified for secretory functions. The full
extent of their functions is still unclear. Macrophages in granulomas are
commonly further modified to form multinucleate giant cells. These arise
by fusion of epithelioid macrophages without nuclear or cellular division
forming huge single cells which may contain dozens of nuclei. In some
circumstances the nuclei are arranged round the periphery of the cell,
termed a Langhans-type giant cell; in other circumstances the nuclei are
randomly scattered throughout the cytoplasm (i.e., the foreign body type
of giant cell which is formed in response to the presence of other
indigestible foreign material in the tissue). Areas of granulomatous
inflammation commonly undergo necrosis.

[0108]Formation of granulomatous inflammation seems to require the
presence of indigestible foreign material (derived from bacteria or other
sources) and/or a cell-mediated immune reaction against the injurious
agent (type IV hypersensitivity reaction).

[0109]The fatty-acid based, pre-cure-derived biomaterials (e.g., coatings
and stand-alone films) of the present invention comprise a hydrophobic
cross-linked fatty acid-derived biomaterial and optionally one or more
therapeutic agents contained in the fatty-acid based, pre-cure-derived
biomaterial. In addition, the pre-cure-derived biomaterials (e.g.,
coatings and stand-alone films) of the present invention are
bio-absorbable as described herein. The therapeutic agent can be an
active agent as contained in the coating and/or a prodrug that, e.g.,
becomes active once released from the coating. In one embodiment of the
invention, the drug eluting pre-cure-derived biomaterial comprised of a
cross-linked fatty acid, e.g., an omega-3 fatty acid. The cross-linked
fatty acid can be non-polymeric. The source of the omega-3 fatty acid can
be a naturally occurring oil, e.g., a fish oil.

[0110]The hydrophobic pre-cure-derived pre-cure biomaterial coatings and
stand-alone films of the present invention may be formed from an oil
component. The oil component can be either an oil, or an oil composition.
The oil component can be a synthetic oil, or a naturally occurring oil,
such as fish oil, cod liver oil, flaxseed oil, grape seed oil, or other
oils having desired characteristics. One embodiment of the present
invention makes use of a fish oil in part because of the high content of
omega-3 fatty acids. The fish oil can also serve as an anti-adhesion
agent. In addition, the fish oil maintains anti-inflammatory or
non-inflammatory properties as well. The present invention is not limited
to formation of the pre-cure-derived biomaterials with fish oil as the
oil starting material. However, the following description makes reference
to the use of fish oil as one example embodiment. Other naturally
occurring oils can be utilized in accordance with the present invention
as described herein.

[0111]It should be noted that as utilized herein, the term fish oil fatty
acid includes, but is not limited to, omega-3 fatty acid, oil fatty acid,
free fatty acid, monoglycerides, di-glycerides, or triglycerides, esters
of fatty acids, or a combination thereof. The fish oil fatty acid
includes one or more of arachidic acid, gadoleic acid, arachidonic acid,
eicosapentaenoic acid, docosahexaenoic acid or derivatives, analogs and
pharmaceutically acceptable salts thereof.

[0113]The present invention relates to a bio-absorbable medical device
coatings and stand-alone films that can exhibit anti-inflammatory
properties, non-inflammatory properties, and anti-adhesion properties,
and the corresponding method of making. The stand-alone film is generally
formed of a naturally occurring oil, such as a fish oil. In addition, the
oil composition can include a therapeutic agent component, such as a drug
or other bioactive agent. The stand-alone film is implantable in a
patient for short term or long term applications. As implemented herein,
the stand-alone film is a fatty-acid based, pre-cure-derived biomaterial
derived at least in part from a fatty acid compound, wherein the
stand-alone film is prepared in accordance with the methods of the
invention. In accordance with further aspects of the present invention,
the stand-alone film can further include a vitamin E compound forming a
portion of the fatty acid compound.

[0115]In accordance with further aspects of the present invention, the
therapeutic agent is combined with the fatty acid compound prior to
formation of the film, resulting in the therapeutic agent being
interspersed throughout the film. Alternatively, the therapeutic agent is
applied to the film in the form of a coating. In accordance with further
aspects of the present invention, the stand-alone film is bioabsorbable.
The stand-alone film can further maintain anti-adhesive properties.

[0116]In accordance with still another embodiment of the present
invention, a method of forming a stand-alone film is introduced. The
method includes providing a fatty acid compound in liquid form and
applying the fatty acid compound to a substrate. The method also includes
curing the fatty acid compound to form the stand-alone film. In
accordance with one aspect of the present invention, the substrate
includes expanded polytetrafluoroethylene (ePTFE) or
polytetrafluoroethylene (PTFE). In accordance with further aspects of the
present invention, the curing includes using at least one curing method
selected from a group of curing methods including application of UV light
and application of heat. The UV light can also be applied to set the
fatty acid compound by forming a skin on the top surface of the fatty
acid compound in liquid form prior to additional curing. In accordance
with further aspects of the present invention, the substrate has an
indentation that is used as a mold to shape the stand-alone film.
Alternatively, the method can further include the step of cutting the
film to a desirable shape.

[0117]The stand-alone film of the present invention may be used as a
barrier to keep tissues separated to avoid adhesion. Application examples
for adhesion prevention include abdominal surgeries, spinal repair,
orthopedic surgeries, tendon and ligament repairs, gynecological and
pelvic surgeries, and nerve repair applications. The stand-alone film may
be applied over the trauma site or wrapped around the tissue or organ to
limit adhesion formation. The addition of therapeutic agents to the
stand-alone films used in these adhesion prevention applications can be
utilized for additional beneficial effects, such as pain relief or
infection minimization. Other surgical applications of the stand-alone
film may include using a stand-alone film as a dura patch, buttressing
material, internal wound care (such as a graft anastomotic site), and
internal drug delivery system. The stand-alone film may also be used in
applications in transdermal, wound healing, and non-surgical fields. The
stand-alone film may be used in external wound care, such as a treatment
for burns or skin ulcers. The stand-alone film may be used without any
therapeutic agent as a clean, non-permeable, non-adhesive,
non-inflammatory, anti-inflammatory dressing, or the stand-alone film may
be used with one or more therapeutic agents for additional beneficial
effects. The stand-alone film may also be used as a transdermal drug
delivery patch when the stand-alone film is loaded or coated with one or
more therapeutic agents.

Oils

[0118]With regard to the aforementioned oils, it is generally known that
the greater the degree of unsaturation in the fatty acids the lower the
melting point of a fat, and the longer the hydrocarbon chain the higher
the melting point of the fat. A polyunsaturated fat, thus, has a lower
melting point, and a saturated fat has a higher melting point. Those fats
having a lower melting point are more often oils at room temperature.
Those fats having a higher melting point are more often waxes or solids
at room temperature. Therefore, a fat having the physical state of a
liquid at room temperature is an oil. In general, polyunsaturated fats
are liquid oils at room temperature, and saturated fats are waxes or
solids at room temperature.

[0119]Polyunsaturated fats are one of four basic types of fat derived by
the body from food. The other fats include saturated fat, as well as
monounsaturated fat and cholesterol. Polyunsaturated fats can be further
composed of omega-3 fatty acids and omega-6 fatty acids. Under the
convention of naming the unsaturated fatty acid according to the position
of its first double bond of carbons, those fatty acids having their first
double bond at the third carbon atom from the methyl end of the molecule
are referred to as omega-3 fatty acids. Likewise, a first double bond at
the sixth carbon atom is called an omega-6 fatty acid. There can be both
monounsaturated and polyunsaturated omega fatty acids.

[0120]Omega-3 and omega-6 fatty acids are also known as essential fatty
acids because they are important for maintaining good health, despite the
fact that the human body cannot make them on its own. As such, omega-3
and omega-6 fatty acids must be obtained from external sources, such as
food. Omega-3 fatty acids can be further characterized as containing
eicosapentaenoic acid (EPA), docosahexanoic acid (DHA), and
alpha-linolenic acid (ALA). Both EPA and DHA are known to have
anti-inflammatory effects and wound healing effects within the human
body.

[0121]As utilized herein, the term "bio-absorbable" generally refers to
having the property or characteristic of being able to penetrate the
tissue of a patient's body. In certain embodiments of the present
invention bio-absorption occurs through a lipophilic mechanism. The
bio-absorbable substance can be soluble in the phospholipid bi-layer of
cells of body tissue, and therefore impact how the bio-absorbable
substance penetrates into the cells.

[0122]It should be noted that a bio-absorbable substance is different from
a biodegradable substance. Biodegradable is generally defined as capable
of being decomposed by biological agents, or capable of being broken down
by microorganisms or biological processes. Biodegradable substances can
cause inflammatory response due to either the parent substance or those
formed during breakdown, and they may or may not be absorbed by tissues.
Because the materials of the invention are biocompatible, and they
hydrolyze into non-inflammatory components, and are subsequently
bio-absorbed by surrounding tissue, they are referred to as
"biomaterials."

Drug Delivery

[0123]The pre-cure-derived biomaterials (e.g, coatings and stand-alone
films) of the present invention deliver one or more therapeutic agents
locally to a targeted area using a stand-alone film, medical device or
apparatus bearing the coating at a selected targeted tissue location of
the patient that requires treatment. The therapeutic agent is released
from the biomaterial to the targeted tissue location. The local delivery
of a therapeutic agent enables a more concentrated and higher quantity of
therapeutic agent to be delivered directly at the targeted tissue
location, without having broader systemic side effects. With local
delivery, the therapeutic agent that escapes the targeted tissue location
dilutes as it travels to the remainder of the patient's body,
substantially reducing or eliminating systemic side effects.

[0124]Targeted local therapeutic agent delivery using a fatty-acid based,
pre-cure-derived biomaterial (e.g, coatings and stand-alone films) can be
further broken into two categories, namely, short term and long term. The
short term delivery of a therapeutic agent occurs generally within a
matter of seconds or minutes to a few days or weeks. The long term
delivery of a therapeutic agent occurs generally within weeks to months.

[0125]The phrase "sustained release" as used herein generally refers to
the release of a biologically active agent that results in the long term
delivery of the active agent.

[0126]The phrase "controlled release" as used herein generally refers to
the release of a biologically active agent in a substantially predictable
manner over the time period of weeks or months, as desired and
predetermined upon formation of the biologically active agent on the
medical device from which it is being released. Controlled release
includes the provision of an initial burst of release upon implantation,
followed by the substantially predictable release over the aforementioned
time period.

Drug Release Mechanisms

[0127]Prior attempts to create films and drug delivery platforms, such as
in the field of stents, primarily make use of high molecular weight
synthetic polymer based materials to provide the ability to better
control the release of the therapeutic agent. Essentially, the polymer in
the platform releases the drug or agent at a predetermined rate once
implanted at a location within the patient. Regardless of how much of the
therapeutic agent would be most beneficial to the damaged tissue, the
polymer releases the therapeutic agent based on properties of the
polymer, e.g., diffusion in a biostable polymer and bulk erosion in a
biodegradable polymeric material. Accordingly, the effect of the
therapeutic agent is substantially local at the surface of the tissue
making contact with the medical device having the coating. In some
instances the effect of the therapeutic agent is further localized to the
specific locations of, for example, stent struts or mesh pressed against
the tissue location being treated. High concentrations of therapeutic
agent present in tissue adjacent to a polymer that elicits an
inflammatory response can create the potential for a localized toxic
effect.

[0128]In various embodiments of the present invention, the fatty-acid
based, pre-cure-derived biomaterial of the invention (e.g., coatings and
stand-alone films) release one or more therapeutic agents by a
dissolution mechanism, e.g., dissolution of a therapeutic agent contained
in a soluble component of the coating into the medium in contact with the
coating, (e.g., tissue), in addition to an erosion based release
mechanism. As a result, the drug release mechanism can be based on the
solubility of the therapeutic agent in the surrounding medium. For
example, a therapeutic agent near the interface between the hydrophobic
coating and the surrounding medium can experience a chemical potential
gradient that can motivate the therapeutic agent out of the oil based
coating and into solution in the surrounding medium. Accordingly, in
various embodiments, the release of a therapeutic agent is not
rate-limited by the break-down or bulk erosion of the coating.

[0129]In various embodiments, the break-down products of the fatty-acid
based, pre-cure-derived biomaterial of the invention are non-inflammatory
byproducts, e.g., free fatty acids and glycerides, that themselves can
release one or more of the therapeutic agents via a dissolution
mechanism.

[0130]In various embodiments, the fatty-acid based, pre-cure derived
biomaterial breaks-down according to a controlled surface erosion
mechanism, thereby releasing one or more therapeutic agents to the
surrounding medium, e.g., tissue, via a dissolution mechanism.

Therapeutic Agents

[0131]As utilized herein, the phrase "therapeutic agent(s)" refers to a
number of different drugs or agents available, as well as future agents
that may be beneficial for use with the fatty acid-derived, pre-cured
biomaterials (e.g., coatings and stand-alone films) of the present
invention. The therapeutic agent component can take a number of different
forms including anti-oxidants, anti-inflammatory agents, anti-coagulant
agents, drugs to alter lipid metabolism, anti-proliferatives,
anti-neoplastics, tissue growth stimulants, functional protein/factor
delivery agents, anti-infective agents, anti-imaging agents, anesthetic
agents, therapeutic agents, tissue absorption enhancers, anti-adhesion
agents, germicides, anti-septics, analgesics, prodrugs thereof, and any
additional desired therapeutic agents such as those listed in Table 1
below.

[0132]Some specific examples of therapeutic agents useful in the
anti-restenosis realm include cerivastatin, cilostazol, fluvastatin,
lovastatin, paclitaxel, pravastatin, rapamycin, a rapamycin carbohydrate
derivative (for example, as described in U.S. Pat. No. 7,160,867), a
rapamycin derivative (for example, as described in U.S. Pat. No.
6,200,985), everolimus, seco-rapamycin, seco-everolimus, and simvastatin.
With systemic administration, the therapeutic agent is administered
orally or intravenously to be systemically processed by the patient.
However, there are drawbacks to a systemic delivery of a therapeutic
agent, one of which is that the therapeutic agent travels to all portions
of the patient's body and can have undesired effects at areas not
targeted for treatment by the therapeutic agent. Furthermore, large doses
of the therapeutic agent only amplify the undesired effects at non-target
areas. As a result, the amount of therapeutic agent that results in
application to a specific targeted location in a patient may have to be
reduced when administered systemically to reduce complications from
toxicity resulting from a higher dosage of the therapeutic agent.

[0133]The term "mTOR targeting compound" refers to any compound that
modulates mTOR directly or indirectly. An example of an "mTOR targeting
compound" is a compound that binds to FKBP 12 to form, e.g., a complex,
which in turn inhibits phosphoinositide (PI)-3 kinase, that is, mTOR. In
various embodiments, mTOR targeting compounds inhibit mTOR. Suitable mTOR
targeting compounds include, for example, rapamycin and its derivatives,
analogs, prodrugs, esters and pharmaceutically acceptable salts.

[0134]Calcineurin is a serine/threonine phospho-protein phosphatase and is
composed of a catalytic (calcineurin A) and regulatory (calcineurin B)
subunit (about 60 and about 18 kDa, respectively). In mammals, three
distinct genes (A-alpha, A-beta, A-gamma) for the catalytic subunit have
been characterized, each of which can undergo alternative splicing to
yield additional variants. Although mRNA for all three genes appears to
be expressed in most tissues, two isoforms (A-alpha and A-beta) are most
predominant in brain.

[0135]The calcineurin signaling pathway is involved in immune response as
well as apoptosis induction by glutamate excitotoxicity in neuronal
cells. Low enzymatic levels of calcineurin have been associated with
Alzheimers disease. In the heart or in the brain calcineurin also plays a
key role in the stress response after hypoxia or ischemia.

[0136]Substances that are able to block the calcineurin signal pathway can
be suitable therapeutic agents for the present invention. Examples of
such therapeutic agents include, but are not limited to, FK506,
tacrolimus, cyclosporin and include derivatives, analogs, esters,
prodrugs, pharmaceutically acceptably salts thereof, and conjugates
thereof which have or whose metabolic products have the same mechanism of
action. Further examples of cyclosporin derivatives include, but are not
limited to, naturally occurring and non-natural cyclosporins prepared by
total- or semi-synthetic means or by the application of modified culture
techniques. The class comprising cyclosporins includes, for example, the
naturally occurring Cyclosporins A through Z, as well as various
non-natural cyclosporin derivatives, artificial or synthetic cyclosporin
derivatives. Artificial or synthetic cyclosporins can include
dihydrocyclosporins, derivatized cyclosporins, and cyclosporins in which
variant amino acids are incorporated at specific positions within the
peptide sequence, for example, dihydro-cyclosporin D.

[0137]In various embodiments, the therapeutic agent comprises one or more
of a mTOR targeting compound and a calcineurin inhibitor. In various
embodiments, the mTOR targeting compound is a rapamycin or a derivative,
analog, ester, prodrug, pharmaceutically acceptably salts thereof, or
conjugate thereof which has or whose metabolic products have the same
mechanism of action. In various embodiments, the calcineurin inhibitor is
a compound of Tacrolimus, or a derivative, analog, ester, prodrug,
pharmaceutically acceptably salts thereof, or conjugate thereof which has
or whose metabolic products have the same mechanism of action or a
compound of Cyclosporin or a derivative, analog, ester, prodrug,
pharmaceutically acceptably salts thereof, or conjugate thereof which has
or whose metabolic products have the same mechanism of action.

[0139]A therapeutically effective amount refers to that amount of a
compound sufficient to result in amelioration of symptoms, e.g.,
treatment, healing, prevention or amelioration of the relevant medical
condition, or an increase in rate of treatment, healing, prevention or
amelioration of such conditions. When applied to an individual active
ingredient, administered alone, a therapeutically effective amount refers
to that ingredient alone. When applied to a combination, a
therapeutically effective amount can refer to combined amounts of the
active ingredients that result in the therapeutic effect, whether
administered in combination, serially or simultaneously. In various
embodiments, where formulations comprise two or more therapeutic agents,
such formulations can be described as a therapeutically effective amount
of compound A for indication A and a therapeutically effective amount of
compound B for indication B, such descriptions refer to amounts of A that
have a therapeutic effect for indication A, but not necessarily
indication B, and amounts of B that have a therapeutic effect for
indication B, but not necessarily indication A.

[0140]Actual dosage levels of the active ingredients in a fatty-acid
based, pre-cure-derived biomaterial (e.g., coating and stand-alone film)
of the present invention may be varied so as to obtain an amount of the
active ingredients which is effective to achieve the desired therapeutic
response without being unacceptably toxic. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the activity
of the particular therapeutic agent (drug) employed, or the ester, salt
or amide thereof, the mechanism of drug action, the time of
administration, the drug release profile of the coating, the rate of
excretion of the particular compounds being employed, the duration of the
treatment, other drugs, compounds and/or materials used in combination
with the particular compounds employed, and like factors known in the
medical arts. For example, the invention provides a fatty-acid based,
pre-cure-derived biomaterial comprising a partially cross-linked fatty
acid and a therapeutic agent, wherein the therapeutic agent comprises at
least 30%, e.g., at least 40%, e.g., at least 50%, e.g., at least 60%,
e.g., at least 70%, by weight, of the biomaterial composition. In
addition to the therapeutic agent, the biomaterial can include vitamin E
in addition to the therapeutic agent.

Other Agents

[0141]The pre-cure-derived biomaterials (e.g., coatings and stand-alone
films) of the present invention may also comprise one or more other
chemicals and entities in addition to the therapeutic agent, including,
but not limited to, one or more of: a pharmaceutically acceptable
carrier, an excipient, a surfactant, a binding agent, an adjuvant agent,
and/or a stabilizing agent (including preservatives, buffers and
antioxidants). The other agents can perform one or more functions, such
as, e.g., an adjuvant may also serve as a stabilizing agent.

[0142]In various embodiments, the coatings and stand-alone films of the
present invention include one or more of a free radical scavenger and
uptake enhancer. In various embodiments, the coatings and stand-alone
films comprise vitamin E.

[0143]It should be noted that as utilized herein to describe the present
invention, the term vitamin E and the term alpha-tocopherol, are intended
to refer to the same or substantially similar substance, such that they
are interchangeable and the use of one includes an implicit reference to
both. Further included in association with the term vitamin E are such
variations including but not limited to one or more of alpha-tocopherol,
beta-tocopherol, delta-tocopherol, gamma-tocopherol, alpha-tocotrienol,
beta-tocotrienol, delta-tocotrienol, gamma-tocotrienol, alpha-tocopherol
acetate, beta-tocopherol acetate, gamma-tocopherol acetate,
delta-tocopherol acetate, alpha-tocotrienol acetate, beta-tocotrienol
acetate, delta-tocotrienol acetate, gamma-tocotrienol acetate,
alpha-tocopherol succinate, beta-tocopherol succinate, gamma-tocopherol
succinate, delta-tocopherol succinate, alpha-tocotrienol succinate,
beta-tocotrienol succinate, delta-tocotrienol succinate,
gamma-tocotrienol succinate, mixed tocopherols, vitamin E TPGS,
derivatives, analogs and pharmaceutically acceptable salts thereof.

[0144]Compounds that move too rapidly through a tissue may not be
effective in providing a sufficiently concentrated dose in a region of
interest. Conversely, compounds that do not migrate in a tissue may never
reach the region of interest. Cellular uptake enhancers such as fatty
acids and cellular uptake inhibitors such as alpha-tocopherol can be used
alone or in combination to provide an effective transport of a given
compound to a given region or location. Both fatty acids and
alpha-tocopherol can be included in the fatty acid-derived, pre-cured
biomaterials (e.g., coatings and stand-alone films) of the present
invention described herein. Accordingly, fatty acids and alpha-tocopherol
can be combined in differing amounts and ratios to contribute to an fatty
acid-derived, pre-cured biomaterial (e.g., coating and stand-alone film)
in a manner that provides control over the cellular uptake
characteristics of the coating and any therapeutic agents mixed therein.

[0145]For example, the amount of alpha-tocopherol can be varied in the
coating. Alpha-tocopherol is known to slow autoxidation in fish oil by
reducing hydroperoxide formation, which results in a decrease in the
amount of cross-linking in a cured fatty acid-derived, pre-cured
biomaterial. In addition, alpha-tocopherol can be used to increase
solubility of drugs in the oil forming the coating. In various
embodiments, alpha-tocopherol can actually protect the therapeutic drug
during curing, which increases the resulting drug load in the coating
after curing. Furthermore, with certain therapeutic drugs, the increase
of alpha-tocopherol in the coating can serve to slow and extend drug
release due to the increased solubility of the drug in the
alpha-tocopherol component of the coating. This reflects the cellular
uptake inhibitor functionality of alpha-tocopherol, in that the uptake of
the drug is slowed and extended over time.

Curing and the Formation of Pre-Cures and Fatty-Acid-Based, Pre-Cure
Derived Biomaterials

[0146]Several methods are available to cure the oil starting material to
form the pre-cure, and then to cure the pre-cure (optionally containing
one or more therapeutic agents) to produce a fatty-acid-based,
pre-cure-derived biomaterial for a drug release and delivery coating or
stand-alone film in accordance with the present invention (for example,
as described in US Patent Application Publications 2008/0118550,
2007/0202149, 2007/0071798, 2006/0110457, 2006/0078586, 2006/0067983,
2006/0067976, 2006/0067975). Preferred methods for curing the starting
material to produce a pre-cure, and then a fatty-acid-based,
pre-cure-derived biomaterial include, but are not limited to, heating
(e.g., employing an oven, a broadband infrared (IR) light source, a
coherent IR light source (e.g., laser), and combinations thereof) and
ultraviolet (UV) irradiation. The starting material may be cross-linked
through auto-oxidation (i.e., oxidative cross-linking).

[0147]In accordance with various embodiments described herein, the drug
release coatings of the present invention are formed of a
pre-cure-derived biomaterial, which can be derived from saturated and
unsaturated fatty acid compounds (e.g., free fatty acids, fatty acid
ester, monoglycerides, diglycerides, triglycerides, metal salts, etc.).
Preferably, the source of fatty acids described herein is saturated and
unsaturated fatty acids such as those readily available in triglyceride
form in various oils (e.g., fish oils). One method of the formation of a
pre-cure-derived biomaterial is accomplished through autoxidation of the
oil. As a liquid oil containing unsaturated fatty acid is heated,
autoxidation occurs with the absorption of oxygen into the oil to create
hydroperoxides in an amount dependent upon the amount of unsaturated
(C═C) sites in the oil. However, the (C═C) bonds are not consumed
in this initial reaction. Concurrent with the formation of hydroperoxides
is the isomerization of (C═C) double bonds from cis to trans in
addition to double bond conjugation. Continued heating of the oil results
in the solidifying of the coating through the formation of cross-linking
and by the further reaction of the hydroperoxides and the cleavage of
C═C double bonds, which convert them into lower molecular weight
secondary oxidation byproducts including aldehydes, ketones, alcohols,
fatty acids, esters, lactones, ethers, and hydrocarbons which can either
remain within the coating and/or are volatilized during the process.

[0148]The type and amount of cross-links formed during oil oxidation can
be tailored depending on the conditions selected (e.g., coating
thickness, temperature, metal composition, etc.). For instance, heating
of the oil allows for cross-linking between the fish oil unsaturated
chains using a combination of peroxide (C--O--O--C), ether (C--O--C), and
hydrocarbon (C--C) bridges (see, e.g., F. D. Gunstone, "Fatty Acid and
Lipid Chemistry." 1999). However, heating at lower temperatures (i.e.,
below 150° C.) results in the formation of predominantly peroxide
cross-links where heating at higher temperatures (i.e., above 150°
C.) results in the thermal degradation of peroxides and C═C and ether
cross-links dominate (F. D. Gunstone, 1999). Schematic illustrations of
various cross-linking mechanisms and schemes are shown in FIGS. 1-2.

[0149]In addition to thermal curing processes, oxidation of oils can also
be induced by light (e.g., photo-oxygenation). Photo-oxygenation is
limited to C═C carbon atoms and results in a conversion from cis to
trans C═C isomers during curing (as occurs with heat initiated
curing). However, photo-oxygenation using UV is a relatively quicker
reaction than autoxidation from heat curing, in the realm of about
1000-1500 times faster. The quicker reaction especially holds true for
methylene interrupted polyunsaturated fatty acids, such as EPA and DHA,
which are found in the fish oil based embodiments of the present
invention.

[0150]An important aspect of UV curing when compared to heat curing is
that although the byproducts obtained by both curing methods are similar,
they are not necessarily identical in amount or chemical structure. One
reason for this is due to the ability of photo-oxygenation to create
hydroperoxides at more possible C═C sites.

[0151]Photo-oxygenation, such as that which results from UV curing, due to
its enhanced ability to create inner hydroperoxides, also results in the
ability to form relatively greater amounts of cyclic byproducts, which
also relates to peroxide cross-linking between fish oil hydrocarbon
chains. For example, photo-oxygenation of linolenate results in 6
different types of hydroperoxides to be formed, whereas autoxidation
results in only 4. The greater amount of hydroperoxides created using
photo-oxygenation results in a similar, but slightly different, structure
and amount of secondary byproducts to be formed relative to autoxidation
from heat curing. Specifically, these byproducts are aldehydes, ketones,
alcohols, fatty acids, esters, lactones, ethers, and hydrocarbons.

[0152]Depending on the oil curing conditions and the fatty acid
composition of the starting oil, a fatty acid-derived biomaterial (i.e.,
pre-cure and pre-cure derived) can be produced by curing the oil so as to
oxidize the double bonds of the unsaturated fatty acid chains while
predominantly preserving triglyceride ester functional groups. The
oxidation of the unsaturated fatty acid chains results in the formation
of hydroperoxides, which, with continued curing, are converted into
aldehydes, ketones, alcohols, fatty acids, esters, lactones, ethers, and
hydrocarbons. With continued heating of the oxidized oil, the byproducts
are volatilized, resulting in an increase in the coating viscosity in
addition to the formation of ester cross-links. The formation of ester
and lactone cross-links can occur different types of mechanisms (i.e.,
esterification, alcoholysis, acidolysis, interesterification as described
in F. D. Gunstone, 1999) between the hydroxyl and carboxyl functional
components in the coating formed from the oxidation process (i.e.,
glyceride and fatty acid). The cross-linking reaction can form different
types of ester linkages such as ester, anhydride, aliphatic peroxide, and
lactones. FIGS. 3-4 summarize the mechanism for the formation of the oil
derived biomaterial and reaction chemistry, respectively. As described in
FIG. 3, after oxidation of the oil, i.e., after forming the precure, a
therapeutic agent can optionally be added. Vitamin E can also be added in
addition to the therapeutic agent, which will protect the agent and
pre-cured oil from further oxidation, but does not inhibit further cross
linking of the fatty acid and/or glyceride components of the oil. FIG. 5
provides a schematic of different methods to form esters from oils
reaction schemes for illustrative purposes, but is not meant to be
limiting in its scope to the invention.

[0153]Pre-cure-derived biomaterial coatings and stand-alone films of the
present invention can be formed from an oil component. The term "oil
component" is also referred to herein as the "oil-containing starting
material." The "oil-containing starting material" may be natural or
derived from synthetic sources. Preferably, the "oil containing starting
material" comprises unsaturated fatty acids. The oil component can be
either an oil, or an oil composition. The oil component can be a
naturally occurring oil, such as fish oil, flax seed oil, grape seed oil,
a synthetic oil, or other oils having desired characteristics. One
example embodiment of the present invention makes use of a fish oil in
part because of the high content of omega-3 fatty acids, which can
provide healing support for damaged tissue, as discussed herein. The fish
oil can also serve as an anti-adhesion agent. In addition, the fish oil
maintains anti-inflammatory or non-inflammatory properties as well. The
present invention is not limited to formation of the fatty acid-derived,
pre-cured biomaterial with fish oil as the naturally occurring oil.
However, the following description makes reference to the use of fish oil
as one example embodiment. Other naturally occurring oils or synthetic
oils can be utilized in accordance with the present invention as
described herein.

[0154]Biodegradable and bioabsorbable implantable materials with ester,
lactone, and anhydride functional groups are typically broken down by
either chemical and/or enzymatic hydrolysis mechanisms (K. Park et al.,
"Biodegradable Hydrogels for Drug Delivery." 1993; J. M. Andersen,
"Perspectives on the In-Vivo Responses of Biodegradable Polymers." in
Biomedical Applications of Synthetic Biodegradable Polymers, edited by
Jeffrey O. Hollinger. 1995, pgs 223-233). Chemical hydrolysis of a
pre-cure-derived biomaterial occurs when the functional group present in
the material is cleaved by water. An example of chemical hydrolysis of a
triglyceride under basic conditions is presented in FIG. 6. Enzymatic
hydrolysis is the cleavage of functional groups in a pre-cure-derived
biomaterial caused by the reaction with a specific enzyme (i.e.,
triglycerides are broken down by lipases (enzymes) that result in free
fatty acids that can then be transported across cell membranes). The
length of time a biodegradable and/or biodegradable pre-cure-derived
biomaterial takes to be hydrolyzed is dependent on several factors such
as the cross-linking density of the material, the thickness, the
hydration ability of the coating, the crystallinity of the
pre-cure-derived biomaterial, and the ability for the hydrolysis products
to be metabolized by the body (K. Park et al., 1993 and J. M. Andersen,
1995).

[0155]A bio-absorbable substance is different from a biodegradable
substance. Biodegradable is generally defined as capable of being
decomposed by biological agents, or capable of being broken down by
microorganisms or biological processes. Biodegradable substances can
cause an inflammatory response due to either the parent substance or
those formed during breakdown, and they may or may not be absorbed by
tissues. Some biodegradable substances are limited to bulk erosion
mechanism for breakdown. For example, a commonly used biodegradable
polymer, PLGA (poly(lactic-co-glycolic acid)) undergoes chemical
hydrolysis in-vivo to form two alpha-hydroxy acids, specifically glycolic
and lactic acids. Although glycolic and lactic acids are byproducts of
various metabolic pathways in the body, it has been previously
demonstrated in previous medical implant and local drug delivery
applications that a local concentration of these products results in an
acidic environment to be produced, which can lead to inflammation and
damage to local tissue (S. Dumitriu, "Polymeric Biomaterials." 2002).
Clinically, this can lead to impaired clinical outcomes such as
restenosis (D. E. Drachman and D. I. Simon. Current Atherosclerosis
Reports. 2005, Vol 7, pgs 44-49; S. E. Goldblum et al. Infection and
Immunity. 1989, Vol. 57, No. 4, pgs 1218-1226) and impaired healing in a
coronary stent application which can lead to late-stent thrombosis or
adhesion formation in an abdominal hernia repair (Y. C. Cheong et al.
Human Reproduction Update. 2001; Vol. 7, No. 6: pgs 556-566). Thus, an
ideal pre-cure-derived biomaterial should not only demonstrate excellent
biocompatibility upon implantation, but should also maintain that
biocompatibility during the life of its implantation with its hydrolysis
byproducts being absorbable by local tissue.

[0156]The bio-absorbable nature of the pre-cure-derived biomaterials used
as a stand-alone film, a coating for a medical device, or in drug
delivery applications results in the biomaterial being absorbed over time
by the cells of the body tissue. In various embodiments, there are
substantially no substances in the coating, or in vivo conversion
by-products of the coating, that induce an inflammatory response, e.g.,
the coating converts in vivo into non-inflammatory components. For
example, in various embodiments, the coatings of the present invention
upon absorption and hydrolysis do not produce lactic acid and glycolic
acid break-down products in measurable amounts. The chemistry of the
pre-cure-derived biomaterial described herein consists of predominantly
fatty acid and glyceride components that can either be hydrolyzed in-vivo
by chemical and/or enzymatic means which results in the release of fatty
acid and glyceride components that can be transported across cell
membranes. Subsequently, the fatty acid and glyceride components eluted
from the pre-cure-derived biomaterial are directly metabolized by cells
(i.e., they are bio-absorbable). The bio-absorbable nature of the coating
and stand-alone film of the present invention results in the coating
being absorbed over time, leaving only an underlying delivery or other
medical device structure that is biocompatible. There is substantially no
foreign body inflammatory response to the bio-absorbable coating or its
hydrolysis breakdown products in the preferred embodiments of the present
invention.

[0157]The process of making the pre-cure-derived biomaterials (e.g.,
coating or stand-alone film) as described herein led to some unexpected
chemical processes and characteristics in view of traditional scientific
reports in the literature about the oxidation of oils (J. Dubois et al.
JAOCS. 1996, Vol. 73, No. 6., pgs 787-794.H. Ohkawa et al., Analytical
Biochemistry, 1979, Vol. 95, pgs 351-358.; H. H. Draper, 2000, Vol. 29,
No. 11, pgs 1071-1077). Oil oxidation has traditionally been of concern
for oil curing procedures due to the formation of reactive byproducts
such as hydroperoxides and alpha-beta unsaturated aldehydes that are not
considered to be biocompatible (H. C. Yeo et al. Methods in Enzymology.
1999, Vol. 300, pgs 70-78.; S-S. Kim et al. Lipids. 1999, Vol. 34, No. 5,
pgs 489-496). However, the oxidation of fatty acids from oils and fats
are normal and important in the control of biochemical processes in-vivo.
For example, the regulation of certain biochemical pathways, such as to
promote or reduce inflammation, is controlled by different lipid
oxidation products (V. N. Bochkov and N. Leitinger. J. Mol. Med. 2003;
Vol. 81, pgs 613-626). Additionally, omega-3 fatty acids are known to be
important for human health and specifically EPA and DHA are known to have
anti-inflammatory properties in-vivo. However, EPA and DHA are not
anti-inflammatory themselves, but it is the oxidative byproducts they are
biochemically converted into that produce anti-inflammatory effects
in-vivo (V. N. Bochkov and N. Leitinger, 2003; L. J. Roberts II et al.
The Journal of Biological Chemistry. 1998; Vol. 273, No. 22, pgs
13605-13612). Thus, although there are certain oil oxidation products
that are not biocompatible, there are also several others that have
positive biochemical properties in-vivo (V. N. Bochkov and N. Leitinger,
2003; F. M. Sacks and H. Campos. J Clin Endocrinol Metab. 2006; Vol. 91,
No. 2, pgs 398-400; A. Mishra et al. Arterioscler Thromb Vasc Biol. 2004;
pgs 1621-1627). Thus, by selecting the appropriate process conditions, a
fatty acid-derived cross-linked hydrophobic fatty acid-derived, pre-cured
biomaterial (from, e.g., fish oil) can be created and controlled using
oil oxidation chemistry with a final chemical profile that will have a
favorable biological performance in-vivo.

[0158]The process of making a pre-cure-derived biomaterial as described
herein leads to a final chemical profile that is biocompatible, minimizes
adhesion formation, acts as a tissue separating barrier, and is
non-inflammatory with respect to the material chemistry and the products
produced upon hydrolysis and absorption by the body in-vivo. The reason
for these properties is due to several unique characteristics of the
fatty acid-derived, pre-cured biomaterials (e.g., coatings or stand-alone
films) of the invention.

[0159]One important aspect of the invention is that no toxic,
short-chained cross-linking agents (such as glutaraldehyde) are used to
form the fatty acid-derived, pre-cured biomaterials (e.g., coatings or
stand-alone films) of the invention. It has been previously demonstrated
in the literature that short chain cross-linking agents can elute during
hydrolysis of biodegradable polymers and cause local tissue inflammation.
The process of creating pre-cure-derived biomaterials does not involve
cross-linking agents because the oil is solely cured into a coating using
oil autoxidation or photo-oxidation chemistry. The oxidation process
results in the formation of carboxyl and hydroxyl functional groups that
allow for the pre-cure-derived biomaterial to become hydrated very
rapidly and become slippery, which allows for frictional injury during
and after implantation to be significantly reduced and/or eliminated. The
methods of making the pre-cure-derived biomaterials described herein
allow the alkyl chains of the fatty acid, glyceride and other lipid
byproducts present in the coating to be disordered, which creates a
coating that is flexible and aids in handling of the material while being
implanted.

[0160]There are several individual chemical components of the coating that
aid in its biocompatibility and its low to non-inflammatory response
observed in-vivo. One critical aspect is that the process of creating a
pre-cure-derived biomaterial as described herein results in low to
non-detectable amounts of oxidized lipid byproducts of biocompatibility
concern, such as aldehydes. These products are either almost completely
reacted or volatilized during the curing process as described herein. The
process of creating a pre-cure-derived biomaterial largely preserves the
esters of the native oil triglycerides and forms ester and/or lactone
cross-links, which are biocompatible (K. Park et al., 1993; J. M.
Andersen, 1995).

[0161]In addition to general chemical properties of a pre-cure-derived
biomaterial that assists in its biocompatibility, there are also specific
chemical components that have positive biological properties. Another
aspect is that the fatty acid chemistry produced upon creation of a
pre-cure-derived biomaterial is similar to the fatty acid chemistry of
tissue, as presented in FIG. 7. Thus, as fatty acids are eluting from the
coating they are not viewed as being "foreign" by the body and cause an
inflammatory response. In fact, C14 (myristic) and C16 (palmitic) fatty
acids present in the coating have been shown in the literature to reduce
production of α-TNF, an inflammatory cytokine. The expression of
α-TNF has been identified as one of the key cytokines responsible
for "turning on" inflammation in the peritoneal after hernia repair,
which can then lead to abnormal healing and adhesion formation (Y. C.
Cheong et al., 2001). α-TNF is also an important cytokine in
vascular injury and inflammation (D. E. Drachman and D. I. Simon, 2005;
S. E. Goldblum, 1989), such as vascular injury caused during a stent
deployment. In addition to the fatty acids just specified, there have
also been additional oxidized fatty acids identified that have
anti-inflammatory properties. A final component identified from the fatty
acid-derived coatings as described herein are delta-lactones (i.e.,
6-membered ring cyclic esters). Delta-lactones have been identified as
having anti-tumor properties (H. Tanaka et al. Life Sciences 2007; Vol.
80, pgs 1851-1855).

[0162]These components identified are not meant to be limiting in scope to
the present invention as changes in starting oil composition and/or
process conditions can invariably alter the fatty acid and/or oxidative
byproduct profiles and can be tailored as needed depending on the
intended purpose and site of application of the fatty acid-derived,
pre-cured biomaterial.

[0163]In summary, the biocompatibility and observed in in-vivo performance
of pre-cure-derived biomaterials described herein is due to the elution
of fatty acids during hydrolysis of the material during implantation and
healing and is not only beneficial as to prevent a foreign body response
in-vivo due to the similarity of the fatty acid composition of the
material to native tissue (i.e., a biological "stealth" coating), but the
specific fatty acids and/or other lipid oxidation components eluting from
the coating aid in preventing foreign body reactions and reducing or
eliminating inflammation, which leads to improved patient outcomes.
Additionally, the fatty acid and glyceride components eluted from the
pre-cure-derived biomaterial are able to be absorbed by local tissue and
metabolized by cells, in, for example, the Citric Acid Cycle (M. J.
Campell, "Biochemistry: Second Edition." 1995, pgs 366-389). Hence, the
pre-cure-derived biomaterial (e.g., coating or stand-alone film)
described herein is also bioabsorbable.

[0164]Accordingly, in one aspect, the invention provides a bio-absorbable,
oil-based coating for a medical device, comprising a cross-linked fatty
acid oil-derived biomaterial with a pre-cured component and a therapeutic
agent. The invention also provides a bio-absorbable, oil-based
stand-alone film, comprising a cross-linked fatty acid oil-derived
biomaterial with a pre-cured component and a therapeutic agent. The
coating and stand-alone film can be prepared according to the methods
discussed herein.

Methods of Treatment Using Fatty Acid-Derived Materials

[0165]Also provided herein is a fatty acid-based, pre-cure-derived
biomaterial suitable for treating or preventing disorders related to
vascular injury and/or vascular inflammation. The fatty acid-based,
pre-cure-derived biomaterial can also be used to treat or prevent injury
to tissue, e.g., soft tissue. The fatty acid-based, pre-cure-derived
biomaterial can be a coating for a medical device or a stand-alone film.
In another embodiment, the source of the fatty acid for the biomaterial
is an oil, such as fish oil.

[0166]In general, four types of soft tissue are present in humans:
epithelial tissue, e.g., the skin and the lining of the vessels and many
organs; connective tissue, e.g., tendons, ligaments, cartilage, fat,
blood vessels, and bone; muscle, e.g., skeletal (striated), cardiac, or
smooth; and nervous tissue, e.g., brain, spinal cord and nerves. The
fatty acid-based, pre-cure-derived biomaterial of the invention (e.g.,
pre-cure-derived stand-alone film) can be used to treat injury to these
soft tissue areas. Thus, in one embodiment, the fatty acid-based,
pre-cure-derived biomaterial of the invention (e.g., pre-cured
stand-alone film) can be used for promotion of proliferation of soft
tissue for wound healing. Furthermore, following acute trauma, soft
tissue can undergo changes and adaptations as a result of healing and the
rehabilitative process. Such changes include, but are not limited to,
metaplasia, which is conversion of one kind of tissue into a form that is
not normal for that tissue; dysplasia, with is the abnormal development
of tissue; hyperplasia, which is excessive proliferation of normal cells
in the normal tissue arrangement; and atrophy, which is a decrease in the
size of tissue due to cell death and resorption or decreased cell
proliferation. Accordingly, the fatty acid-based, pre-cure-derived
biomaterial of the invention (e.g., pre-cured stand-alone film) can be
used for the diminishment or alleviation of at least one symptom
associated with or caused by acute trauma in soft tissue.

[0167]In one embodiment of the present invention, as described below, the
fatty acid-based, pre-cure-derived biomaterial can be used, for example,
to prevent tissue adhesion. The tissue adhesion can be, for example, a
result of blunt dissection. Blunt dissection can be generally described
as dissection accomplished by separating tissues along natural cleavage
lines without cutting. Blunt dissection is executed using a number of
different blunt surgical tools, as is understood by those of ordinary
skill in the art. Blunt dissection is often performed in cardiovascular,
colo-rectal, urology, gynecology, upper GI, and plastic surgery
applications, among others.

[0168]After the blunt dissection separates the desired tissues into
separate areas, there is often a need to maintain the separation of those
tissues. In fact, post surgical adhesions can occur following almost any
type of surgery, resulting in serious postoperative complications. The
formation of surgical adhesions is a complex inflammatory process in
which tissues that normally remain separated in the body come into
physical contact with one another and attach to each other as a result of
surgical trauma.

[0169]It is believed that adhesions are formed when bleeding and leakage
of plasma proteins from damaged tissue deposit in the abdominal cavity
and form what is called a fibrinous exudate. Fibrin, which restores
injured tissues, is sticky, so the fibrinous exudate may attach to
adjacent anatomical structures in the abdomen. Post-traumatic or
continuous inflammation exaggerates this process, as fibrin deposition is
a uniform host response to local inflammation. This attachment seems to
be reversible during the first few days after injury because the
fibrinous exudates go through enzymatic degradation caused by the release
of fibrinolytic factors, most notably tissue-type plasminogen activator
(t-PA). There is constant play between t-PA and plasminogen-activator
inhibitors. Surgical trauma usually decreases t-PA activity and increases
plasminogen-activator inhibitors. When this happens, the fibrin in the
fibrinous exudate is replaced by collagen. Blood vessels begin to form,
which leads to the development of an adhesion. Once this has occurred,
the adhesion is believed to be irreversible. Therefore, the balance
between fibrin deposition and degradation during the first few days
post-trauma is critical to the development of adhesions (Holmdahl L.
Lancet 1999; 353: 1456-57). If normal fibrinolytic activity can be
maintained or quickly restored, fibrous deposits are lysed and permanent
adhesions can be avoided. Adhesions can appear as thin sheets of tissue
or as thick fibrous bands.

[0170]Often, the inflammatory response is also triggered by a foreign
substance in vivo, such as an implanted medical device. The body sees
this implant as a foreign substance, and the inflammatory response is a
cellular reaction to wall off the foreign material. This inflammation can
lead to adhesion formation to the implanted device; therefore a material
that causes little to no inflammatory response is desired.

[0171]Thus, the fatty acid-based, pre-cure-derived biomaterial (e.g.,
stand-alone film) of the present invention may be used as a barrier to
keep tissues separated to avoid the formation of adhesions, e.g.,
surgical adhesions. Application examples for adhesion prevention include
abdominal surgeries, spinal repair, orthopedic surgeries, tendon and
ligament repairs, gynecological and pelvic surgeries, and nerve repair
applications. The fatty acid-based, pre-cure-derived biomaterial (e.g.,
stand-alone film) may be applied over the trauma site or wrapped around
the tissue or organ to limit adhesion formation. The addition of
therapeutic agents to the fatty acid-based, pre-cure-derived biomaterial
used in these adhesion prevention applications can be utilized for
additional beneficial effects, such as pain relief or infection
minimization. Other surgical applications of the fatty acid-based,
pre-cure-derived biomaterial may include using a stand-alone film as a
dura patch, buttressing material, internal wound care (such as a graft
anastomotic site), and internal drug delivery system. The fatty
acid-based, pre-cure-derived biomaterial may also be used in applications
in transdermal, wound healing, and non-surgical fields. The fatty
acid-based, pre-cure-derived biomaterial may be used in external wound
care, such as a treatment for burns or skin ulcers. The fatty acid-based,
pre-cure-derived biomaterial may be used without any therapeutic agent as
a clean, non-permeable, non-adhesive, non-inflammatory, anti-inflammatory
dressing, or the fatty acid-based, pre-cure-derived biomaterial may be
used with one or more therapeutic agents for additional beneficial
effects. The fatty acid-based, pre-cure-derived biomaterial may also be
used as a transdermal drug delivery patch when the fatty acid-based,
pre-cure-derived biomaterial is loaded or coated with one or more
therapeutic agents.

[0172]The process of wound healing involves tissue repair in response to
injury and it encompasses many different biologic processes, including
epithelial growth and differentiation, fibrous tissue production and
function, angiogenesis, and inflammation. Accordingly, the fatty
acid-based, pre-cure-derived biomaterial (e.g., stand-alone film)
provides an excellent material suitable for wound healing applications.

Modulated Healing

[0173]Also provided herein is a fatty acid-based, pre-cure-derived
biomaterial suitable for achieving modulated healing in a tissue region
in need thereof, wherein the composition is administered in an amount
sufficient to achieve said modulated healing. In one embodiment, the
fatty acid-based, pre-cure-derived biomaterial is a medical coating for a
medical device or a stand-alone film. In another embodiment, the source
of the fatty acid for the biomaterial is an oil, such as fish oil.

[0174]Modulated healing can be described as the in-vivo effect observed
post-implant in which the biological response is altered resulting in a
significant reduction in foreign body response. As utilized herein, the
phrase "modulated healing" and variants of this language generally refers
to the modulation (e.g., alteration, delay, retardation, reduction,
detaining) of a process involving different cascades or sequences of
naturally occurring tissue repair in response to localized tissue injury,
substantially reducing their inflammatory effect. Modulated healing
encompasses many different biologic processes, including epithelial
growth, fibrin deposition, platelet activation and attachment,
inhibition, proliferation and/or differentiation, connective fibrous
tissue production and function, angiogenesis, and several stages of acute
and/or chronic inflammation, and their interplay with each other. For
example, the fatty acids described herein can alter, delay, retard,
reduce, and/or detain one or more of the phases associated with healing
of vascular injury caused by medical procedures, including, but not
limited to, the inflammatory phase (e.g., platelet or fibrin deposition),
and the proliferative phase. In one embodiment, "modulated healing"
refers to the ability of a fatty acid derived biomaterial to alter a
substantial inflammatory phase (e.g., platelet or fibrin deposition) at
the beginning of the tissue healing process. As used herein, the phrase
"alter a substantial inflammatory phase" refers to the ability of the
fatty acid derived biomaterial to substantially reduce the inflammatory
response at an injury site. In such an instance, a minor amount of
inflammation may ensue in response to tissue injury, but this level of
inflammation response, e.g., platelet and/or fibrin deposition, is
substantially reduced when compared to inflammation that takes place in
the absence of the fatty acid derived biomaterial.

[0175]For example, the fatty acid-based, pre-cure-derived biomaterial
(e.g., fatty acid-based, pre-cure-derived coating or fatty acid-based,
pre-cure-derived stand-alone film) of the present invention has been
shown experimentally in animal models to delay or alter the inflammatory
response associated with vascular injury, as well as excessive formation
of connective fibrous tissue following tissue injury. The fatty
acid-based, pre-cure-derived biomaterial (e.g., coating or stand-alone
film) of the present invention can delay or reduce fibrin deposition and
platelet attachment to a blood contact surface following vascular injury.

[0176]Accordingly, the fatty acid-based, pre-cure-derived biomaterial
(e.g., coating or stand-alone film) of the present invention provides an
excellent absorbable cellular interface suitable for use with a surgical
instrument or medical device that results in a modulated healing effect,
avoiding the generation of scar tissue and promoting the formation of
healthy tissue at a modulated or delayed period in time following the
injury. Without being bound by theory, this modulated healing effect can
be attributed to the modulation (e.g., alteration, delay, retardation,
reduction, detaining) of any of the molecular processes associated with
the healing processes of vascular injury. For example, the fatty
acid-based, pre-cure-derived biomaterial (e.g., fatty acid-based,
pre-cure-derived coating or fatty acid-based, pre-cure-derived film) of
the present invention can act as a barrier or blocking layer between a
medical device implant (e.g., a surgical mesh, graft, or stent), or
surgical instrument, and the cells and proteins that compose the vessel
wall, such as the endothelial cells and smooth muscle cells that line the
vessel's interior surface. The barrier layer prevents the interaction
between the surgical implant and the vessel surface, thereby preventing
the initiation of the healing process by the cells and proteins of the
vessel wall. In this respect, the barrier layer acts as a patch that
binds to the vessel wall and blocks cells and proteins of the vessel wall
from recognizing the surgical implant (i.e., the barrier layer blocks
cell-device and/or protein-device interactions), thereby blocking the
initiation of the vascular healing process, and avoiding the fibrin
activation and deposition and platelet activation and deposition.

[0177]In another non-binding example, the modulated healing effect can be
attributed to the modulation (e.g., alteration, delay, retardation,
reduction, detaining) of signaling between the cells and proteins that
compose the vessel wall and various components of the bloodstream that
would otherwise initiate the vascular healing process. Stated
differently, at the site of vascular injury, the fatty acid derived
biomaterial (e.g., coating or stand-alone film) of the present invention
can modulate the interaction of cells of the vessel wall, such as
endothelial cells and/or smooth muscle cells, with other cells and/or
proteins of the blood that would otherwise interact with the damaged
cells to initiate the healing process. Additionally, at the site of
vascular injury, the fatty acid-based, pre-cure-derived biomaterial
(e.g., coating or stand-alone film) of the present invention can modulate
the interaction of proteins of the vessel wall with other cells and/or
proteins of the blood, thereby modulating the healing process.

[0178]The fatty acid-based, pre-cure-derived biomaterial (e.g., coating or
stand-alone film) of the present invention can be designed to maintain
its integrity for a desired period of time, and then begin to hydrolyze
and be absorbed into the tissue that it is surrounded by. Alternatively,
the fatty acid-based, pre-cure-derived biomaterial can be designed such
that, to some degree, it is absorbed into surrounding tissue immediately
after the fatty acid derived biomaterial is inserted in the subject.
Depending on the formulation of the fatty acid-based, pre-cure-derived
biomaterial, it can be completely absorbed into surrounding tissue within
a time period of 1 day to 24 months, e.g., 1 week to 12 months, e.g., 1
month to 10 months, e.g., 3 months to 6 months. Animal studies have shown
resorption of the fatty acid derived biomaterial occurring upon
implantation and continuing over a 3 to 6 month period, and beyond.

Tailoring of Drug Release Profiles

[0179]In various aspects, the present invention provides methods of curing
a of a fatty acid-derived coating, preferably fish oil, to provide a
fatty acid-derived, pre-cured biomaterial coating or stand-alone film
containing one or more therapeutic agents that can tailor the release
profile of a therapeutic agent from the coating or film. The release
profile can be tailored, e.g., through changes in oil (e.g., fish oil)
chemistry by varying coating composition, temperature, and cure times.
The position of the drug-containing layer on the coated device provides
an additional mechanism to alter the release profile of the non-polymeric
cross-linked fatty acid-derived, pre-cured biomaterial coating. This can
be achieved, e.g., by loading a drug into a cured base coating layer and
coating a topcoat overlayer cured coating onto the previously cured
encapsulating base layer.

[0180]An advantage of the cured fish oil coating and stand-alone film in
various embodiments of the present invention is that the curing
conditions utilized (i.e., cure time and temperature) can directly
influence the amount of coating cross-linking density and byproduct
formation, which in turn effects the coating degradation. Thus, by
altering the curing conditions employed, the dissolution rate of a
therapeutic compound of interest contained in the coating can also be
altered.

[0181]In various embodiments of the present invention, an agent, such as,
e.g., a free radical scavenger, can be added to the starting material to
tailor the drug release profile of the fatty acid-derived, pre-cured
biomaterial that is formed. In various embodiments, vitamin E is added to
the starting material to, for example, to slow down autoxidation in fish
oil by reducing hydroperoxide formation, which can result in a decrease
in the amount of cross-linking observed in a cured fish oil coating. In
addition, other agents can be used to increase the solubility of a
therapeutic agent in the oil component of the starting material, protect
the drug from degradation during the curing process, or both. For
example, vitamin E can also be used to increase the solubility of certain
drugs in a fish oil starting material, and thereby facilitate tailoring
the drug load of the eventual cured coating. Thus, varying the amount of
vitamin E present in the coating provides an additional mechanism to
alter the cross-linking and chemical composition of the fatty
acid-derived, pre-cured biomaterials (e.g., coatings and stand-alone
films) of the present invention.

[0182]In various embodiments, the present invention provides coatings and
stand-alone films where the drug release profile of the fatty
acid-derived, pre-cured biomaterial is tailored through the provision of
two or more coatings and selection of the location of the therapeutic
agent. The drug location can be altered, e.g., by coating a bare portion
of a medical device with a first starting material and creating a first
cured coating, then coating at least a portion of the first cured-coating
with the drug-oil formulation to create a second overlayer coating. The
first starting material can contain one or more therapeutic agents. In
various embodiments, the second overlayer coating is also cured. The drug
load, drug release profiles, or both, of the first coating, the overlay
coating, or both, can be tailored through the use of different curing
conditions and/or addition of free radical scavengers (e.g., vitamin E),
as described herein. The process of providing two layers can be extended
to provide three or more layers, wherein at least one of the layers
comprises a hydrophobic, cross-linked fatty acid-derived, pre-cured
biomaterial prepared from a fatty-acid containing oil, such as fish oil.
In addition, one or more of the layers can be drug eluting, and the drug
release profile of such layers can be tailored using the methods
described herein.

[0183]In various embodiments, the present invention provides coatings
where the drug release profile of the overall coating is tailored through
the provision of two or more coating regions with different drug release
profiles and selection of the location of the therapeutic agent. In
various embodiments, the formation of different coating regions with
different drug release properties is obtained by location specific curing
conditions, e.g., location specific UV irradiation, and/or location
specific deposition of a starting material on the coated device, e.g., by
ink jet printing methods.

Coating Approaches

[0184]FIG. 8 illustrates one method of making a medical device of the
present invention, such as, e.g., a drug eluting coated stent, in
accordance with one embodiment of the present invention. The process
involves providing a medical device, such as the stent (step 100). A
coating of a starting material, which is a non-polymeric cross-linked
fatty acid-derived, pre-cured biomaterial coating, is then applied to the
medical device (step 102). One of ordinary skill in the art will
appreciate that this basic method of application of a coating to a
medical device, such as a stent, can have a number of different
variations falling within the process described. The step of applying a
coating substance to form a coating on the medical device can include a
number of different application methods. For example, the medical device
can be dipped into a liquid solution of the coating substance. The
coating substance can be sprayed onto the device. Another application
method is painting the coating substance on to the medical device. One of
ordinary skill in the art will appreciate that other methods, such as
electrostatic adhesion, can be utilized to apply the coating substance to
the medical device. Some application methods may be particular to the
coating substance and/or to the structure of the medical device receiving
the coating. Accordingly, the present invention is not limited to the
specific embodiments of starting material application described herein,
but is intended to apply generally to the application of the starting
material which is to become a fatty acid-derived, pre-cured biomaterial
coating of a medical device, taking whatever precautions are necessary to
make the resulting coating maintain desired characteristics.

[0185]FIG. 9 is a flowchart illustrating one example implementation of the
method of FIG. 8. In accordance with the steps illustrated in FIG. 9, a
bio-absorbable carrier component (e.g., a fatty acid source, such as a
naturally occurring oil) is provided (step 110). The carrier is then
pre-cured ("partially cured") to induce an initial amount of cross
linking (step 112). The resulting material can then be combined with a
therapeutic agent, to form a pre-cured material that is to become a fatty
acid-based, pre-cure-derived biomaterial coating (step 114). The
pre-cured material is applied to the medical device, such as the stent
10, to form the coating (step 116). The coating is then cured (step 118)
by any of the curing methods described herein to form a fatty
acid-derived, pre-cured biomaterial coating.

[0186]In certain instances, a therapeutic agent that is desired for
incorporation into a fatty acid-derived biomaterial coating is not stable
to the thermal/UV curing process utilized to create the device coating
(e.g., there is a significant amount of drug degradation observed). In
order to maintain therapeutic agent composition and minimize degradation
of the therapeutic agent, the fatty acid starting material (e.g., fish
oil) can be first partially cured ("pre-cured"), in the absence of the
therapeutic agent, to oxidize the unsaturated components in the oil. Such
a process increases the viscosity of the oil and reduces its reactivity
by partially cross-linking the fatty acids of the oil, for, e.g., medical
device coating applications. The therapeutic agent can then be combined
with the pre-cure in an organic solvent and sprayed and/or cast onto a
medical device and/or as a stand-alone film material, and subsequently
heated to form a final cross-linked material (i.e., a fatty acid-based,
pre-cure-derived coating) for use in its intended application. This
process results in incorporating the therapeutic agent into the fatty
acid-derived, pre-cured biomaterial for extended drug release from the
coating. Alternatively, after the creation of the pre-cure, an
anti-oxidant such as Vitamin E can be combined with the therapeutic agent
and an organic solvent for application onto a medical device or to create
a stand-alone film. This coating is then also final cured into a fatty
acid-derived, pre-cured biomaterial. While the antioxidant (e.g., Vitamin
E) is oxidized during the final cure step, it prevents the therapeutic
and oil components from being further oxidized and preserves the drug
composition and activity. Although the antioxidant prevents further
oxidation of the therapeutic and pre-cured oil components, it does not
inhibit the formation of ester cross-links between the fatty acids upon
cure, because the reactive carboxyl and hydroxyl functional groups needed
to create the fatty acid-derived, pre-cured biomaterial were created
during the initial thermal/UV curing treatment of the oil starting
material, i.e., during the creation of the pre-cure.

[0187]The coated medical device is then sterilized using any number of
different sterilization processes (step 118). For example, sterilization
can be implemented utilizing ethylene oxide, gamma radiation, E beam,
steam, gas plasma, or vaporized hydrogen peroxide. One of ordinary skill
in the art will appreciate that other sterilization processes can also be
applied, and that those listed herein are merely examples of
sterilization processes that result in a sterilization of the coated
stent, preferably without having a detrimental effect on the coating 20.

[0188]It should be noted that the fatty acid component (e.g., a fish oil)
can be added multiple times to create multiple tiers in forming the
coating. For example, if a thicker coating is desired, additional tiers
of the fatty acid component can be added after steps 100, 102, 110, 112,
114, 116, 118 and/or 120. Different variations relating to when the fatty
acid is cured and when other substances are added are possible in a
number of different process configurations. Accordingly, the present
invention is not limited to the specific sequence illustrated. Rather,
different combinations of the basic steps illustrated are anticipated by
the present invention.

[0189]FIGS. 10A-10E illustrate some of the other forms of medical devices
mentioned above in combination with the coating 10 of the present
invention. FIG. 10A shows a graft 50 with the coating 10 coupled or
adhered thereto. FIG. 10B shows a catheter balloon 52 with the coating 10
coupled or adhered thereto. FIG. 10C shows a stent 54 with the coating 10
coupled or adhered thereto. FIG. 10D illustrates a stent 10 in accordance
with one embodiment of the present invention. The stent 10 is
representative of a medical device that is suitable for having a coating
applied thereon to effect a therapeutic result. The stent 10 is formed of
a series of interconnected struts 12 having gaps 14 formed therebetween.
The stent 10 is generally cylindrically shaped. Accordingly, the stent 10
maintains an interior surface 16 and an exterior surface 18. FIG. 10E
illustrates a coated surgical mesh, represented as a biocompatible mesh
structure 10, in accordance with one embodiment of the present invention.
The biocompatible mesh structure 10 is flexible, to the extent that it
can be placed in a flat, curved, or rolled configuration within a
patient. The biocompatible mesh structure 10 is implantable, for both
short term and long term applications. Depending on the particular
formulation of the biocompatible mesh structure 10, the biocompatible
mesh structure 10 will be present after implantation for a period of
hours to days, or possibly months, or permanently.

[0190]Each of the medical devices illustrated, in addition to others not
specifically illustrated or discussed, can be combined with the coating
10 using the methods described herein, or variations thereof.
Accordingly, the present invention is not limited to the example
embodiments illustrated. Rather the embodiments illustrated are merely
example implementations of the present invention.

[0191]In another embodiment, the biomaterials of the invention, i.e., a
pre-cure biomaterial, or a fatty acid-based, pre-cure-derived biomaterial
can be used in the form of an emulsion. An "emulsion," which is a type of
suspension, is a combination of two or more immiscible liquids in a
particular energetically unstable state. Although an emulsion can be a
combination of more than two immiscible liquids, for the sake of clarity,
the following explanation will be presented assuming an emulsion of only
two liquids. The first liquid is dispersed or suspended in a continuous
phase of the second liquid. This may be thought of as "droplets" of the
first suspended liquid distributed throughout a continuous "pool" of the
second liquid. The first liquid can include a mixture of any number of
miscible liquids and the second liquid can include a mixture of any
number of miscible liquids as long as the mixture of the first liquid is
immiscible with respect to the mixture of the second liquid. One of
ordinary skill in the art will appreciate that emulsions can be
formulated using a combination of three or more immiscible liquids, and
although such embodiments are not described further herein, they are
considered to fall within the scope of the present invention.

[0192]Various aspects and embodiments of the present invention are further
described by way of the following Examples. The Examples are offered by
way of illustration and not by way of limitation.

EXAMPLES

[0193]The following examples characterize the novel fatty acid-derived
biomaterial chemistry described herein and illustrate some of the
boundaries associated with the chemical mechanisms of formation and how
alteration of those mechanisms influences the properties (e.g.,
therapeutic benefits and/or drug release profile) of the final product.
The identity of some of the hydrolysis products are identified through
in-vitro experiments and correlated with in-vivo experiments to
demonstrate the ability for the coating or stand-alone film to be
bioabsorbed. Finally, examples showing the utility of the fatty
acid-derived biomaterials described herein in drug delivery applications
on coronary stents and hernia mesh devices are presented.

[0194]The following examples are for demonstration purposes and are not
meant to be limiting.

[0195]In the following example, coated medical devices (e.g., a
polypropylene mesh) were cured in a high airflow oven at 200° F.
for 24 hours, after which the fish oil was converted into a cross-linked
biomaterial gel coating encapsulating the polypropylene mesh by oxidation
of the C═C bonds present in the fish oil resulting in the formation
of oxidative byproducts (i.e., hydrocarbons, aldehydes, ketones,
glycerides, fatty acids) while largely preserving the esters derived from
the original oil triglycerides. Volatilization of the byproducts followed
by the formation of ester and lactone cross-links result in the
solidification of oil into a bioabsorbable hydrophobic cross-linked
biomaterial. The ability for the coating to be slowly hydrolyzed was
investigated using 0.1 M PBS solution. The PBS solution was analyzed
using GC-FID fatty acid profile and GPC chromatographic measurements
after hydrolysis of the oil-derived biomaterial in PBS for 30 days.

[0196]FIG. 11 summarizes the fatty acid profile results obtained after
drying the PBS solution and then performing a GC-FID fatty acid profile
analysis as described in the AOCS official method Ce 1-89b to identify
the fatty acids present in solution. FIG. 11 shows that the fatty acids
identified from the PBS solution are the same as those detected from the
coating itself. GPC analysis was also conducted on the hydrolysis
solution and the results are summarized in Table 5. The GPC results
showed that the vast majority of molecular weight components identified
(80%) were below a molecular weight of 500, which is consistent with the
fatty acid components of the coating. Also, glyceride components of the
coating could be identified with molecular weights around 1000 (15% of
the coating). The GPC results also showed a negligible amount
(approximately 4%) of high molecular weight gel. The GPC results support
the other analytical characterization experiments on the oil-derived
coatings which show that the oil-derived biomaterial is comprised of
cross-linked glycerides and fatty acids, and that the majority of the
coating is non-polymeric (i.e., approximately 80% of the components
identified had a molecular weight of less than 500).

[0197]In this particular embodiment of the present invention, the
application of cured oil coatings loaded with a therapeutic and applied
to a cardiac stent are presented. The flow diagram presenting the process
to create a cured coating on a stent loaded with a therapeutic is
outlined in FIG. 13. Briefly, a pre-cured fish oil coating is created in
a reaction vessel under agitation with heating in the presence of oxygen
at 200° F. for 20 hours. The coating is mixed with the therapeutic
of interest, and vitamin E with a solvent and then sprayed onto the stent
to create a coating. The coating is annealed to the stent surface by
heating at 200° F. for 7 hours to create a uniform coating. A
coating with a model anti-inflammatory agent showed that this process
allowed for 90% of the drug to be recovered after curing as determined
using extraction of the drug from the device with HPLC analysis. FIG. 14
shows the drug release profile for this coating in 0.01 M PBS buffer
going out to 20 days with over a 90% recovery of the drug using this
process.

Example 3

Controlled Release of the Fatty Acid Based, Pre-Cure Derived Coating

[0198]Drug release was quantified for 3 batches of 16 mm stainless steel
stents, coated with a Compound C drug coating formulation consisting of
60% Compound C, 30% pre-cured Fish Oil, 10% tocopherol Pre-cured fish oil
was produced using fish oil, pre-cured at 93° C., to achieve a
pre-cure viscosity of 1.3×105 cps as measured at 22° C.
The coating was applied to surface of a 16 mm Atrium Flyer stainless
steel stent by spraying to achieve an overall stent drug load of
approximately 100 μg of Compound C per stent. The coated stents were
subjected to a second thermal cure process whereby the coating was
post-cured in an oven at 93° C. for a period of 7.5 hours.
Dissolution was carried out in a 4 ml solution containing 0.01M purified
buffered saline (PBS) at a temperature of 37° C.

[0199]An HPLC method was used to quantify drug dissolution in vitro of the
Compound C drug coated stent. The drug release profile data is shown in
FIG. 15, which illustrates that the drug release from the Compound C drug
coating extends over a period of 20 days and that the release profile is
reproducible from batch to batch.

Example 4

Trackability Forces for Bare Metal, Coated and Drug Coated Stent

[0200]Device trackability forces were quantified for 3.0 mm×13 mm
CoCr stents mounted on a balloon catheter. Trackability forces were
quantified for 3 distinct stent coating groups, a bare metal CoCr stent,
a CoCr stent coated with a 75% pre-cured fish oil, 25% tocopherol coating
and a CoCr stent coated with a 60% Compound B, 22.5% pre-cured fish oil,
12.5% tocopherol coating. Pre-cured fish oil was produced using fish oil,
pre-cured at 93° C., to achieve a pre-cure viscosity of
1.0×106 cps as measured at 22° C. The fatty
acid-derived, pre-cured biomaterial was prepared by dissolving 75%
pre-cured fish oil with 25% tocopherol in MTBE solvent to achieve a 1%
solids formulation. The formulation is vortexed for 1 minute at 3000 RPM.
The formulation is then applied to the stent via a spray process to
achieve a total stent coating weight of approximately 167 μg. The
coated stents are subjected to thermal post-curing at 93° C. for 6
hours. Following the post-curing process, the stents are crimped onto
balloon catheters to achieve a stent profile dimension of approximately
0.04 inches using a 12 point crimping instrument exerting compressive
loads of between 16 and 22 psi. The Compound B, pre-cured fish oil,
tocopherol formulation was prepared by dissolving 75% pre-cured fish oil
with 25% tocopherol in MTBE solvent to achieve a 25% solids formulation.
A quantity of Compound B is weighed out in a proper glass vial and a
volume of the pre-cured FO and tocopherol formulation is added into the
glass vial to achieve a 60% Compound B, 40% pre-cure fish oil-tocopherol
ratio. Additional MTBE is added to the glass vial to achieve a total
solids ratio of 1%. The formulation is vortexed for 1 minute at 3000 rpm.
The formulation is then applied to the stent via a spray process to
achieve a total stent coating weight of approximately 167 μg. The
coated stents are subjected to thermal post-curing at 93° C. for 6
hours. Following the post-curing process, the stents are crimped onto
balloon catheters to achieve a stent profile dimension of approximately
0.04 inches. Catheters with stents mounted over the balloon segment are
then threaded through a 6Fr Medtronic Launcher guide catheter inserted
within a torturous path consisting of an anatomical model containing 2
bends having radii of 22 mm and 14 mm with a total set travel distance of
395 mm. De-Ionized water was used as the test environment. Forces are
measured by a load cell on the mechanism used to drive the catheter
forward.

[0201]Trackability force data is shown in FIG. 16. This data indicates
lower overall trackability forces required to push the devices
incorporating the fatty acid-derived, pre-cured biomaterial coating
containing tocopherol, Compound B and pre-cured fish oil as compared to
the bare metal stent, suggesting that the coatings substantially reduce
the coefficient of friction of the stent surface and subsequently the
frictional forces between the stent and the guide catheter wall during
the process of advancing the balloon catheter within the guide catheter
through a torturous pathway.

Example 5

Post Curing Time and Temperature Affects on Mechanical Properties

[0202]The effects of post curing time and temperature on coating
mechanical properties were evaluated in a study in which CoCr stents were
coated with an oil-derived, pre-cured biomaterial coating of Compound
B/pre-cured fish oil/tocopherol. CoCr stents were pre-cleaned with
acetone, and coated with a formulation of 60% Compound B, 30% pre-cured
fish oil, 10% tocopherol. The Compound B, pre-cured fish oil, tocopherol
formulation was prepared by dissolving 75% pre-cured fish oil with 25%
tocopherol in MTBE solvent to achieve a 25% solids formulation. A
quantity of Compound B is weighed out in a glass vial and a volume of the
pre-cured FO and tocopherol formulation is added into the glass vial to
achieve a 60% Compound B, 40% pre-cure fish oil-tocopherol ratio.
Additional MTBE is added to the glass vial to achieve a total solids
ratio of 1%. The formulation is vortexed for 1 minute at 3000 RPM. The
formulation is then applied to the stent via a spray process to achieve a
total stent coating weight of approximately 167 μg. The coated stents
were then subjected to thermal post-curing process at temperatures
ranging from 60° C. to 100° C.

[0203]Following the post-curing process, the stents are crimped onto
balloon catheters using a twelve point crimping instrument to achieve a
stent profile dimension of approximately 0.043 inches, requiring 16-22
psi of crimping pressure. Subsequently, the balloon catheters were
inflated in air to a nominal inflation pressure of 9 atm. Following
crimping and expansion the drug coating is evaluated visually for
physical damage. The results of this testing demonstrate that stents post
cured at temperatures above 80° C. show substantially less coating
damage following crimping and subsequent expansion than stents post cured
at 80° C. or less, demonstrating that mechanical properties of the
coating can be significantly altered by changing the final cross link
density of the coating by altering the temperature at which the coating
is post cured.

Example 6

Influence of Curing Time and Temperature on Drug Recovery of Thermally
Sensitive Drugs

[0204]The effect of curing time and temperature on drug recovery of a
thermally sensitive drug incorporated within hydrophobic cross linked gel
coating was evaluated and quantified. Pre-cured fish oil (PCFO) was
prepared by heating fish oil in a reactor at 93° C. for a total of
26 hours, while infusing oxygen through a diffuser. The resultant
viscosity of the pre-cured fish oil was 5×106 cps as measured
at 22° C. The formulation consisting of 60% Compound B, 30% PCFO,
and 10% Vitamin E was made by combining 3.76 g PCFO and 1.3 g Vitamin E
to form a 75% PCFO, 25% Vitamin E base coating. 15.04 g
methyl-tert-butyl-ether (MTBE) was added to produce a base coating
solution of 75% solvent, 25% solids. This solution was vortexed for 30
min until clear. Next, 529 mg of the base coating solution was added to
198.8 mg Compound B. This mixture was diluted with 32.8 g
methyl-tert-butyl-ether (MTBE) to produce a final solution of 1.0% solids
for spray coating. CoCr stents (3.0×13 mm) were spray coated using
an ultrasonic spray coating system (SonoTek, Inc.) with a target load of
100 μg Compound B. Each coated stent was weighed before coating and
after coating to determine the actual weight of coating applied to each
stent gravimetrically. Coated stents were subjected to oven post curing
at a temperature range between 60° C. to 100° C. with post
curing time ranging from 0 hrs to 24 hours. Following post curing the
drug coating was extracted in a 100% acetonitrile solution and analyzed
via HPLC to determine the drug concentration in solution from which the
total drug mass extracted from the stent is determined. The total drug
mass extracted from the stents along with the actual coating weight on
the stent determined gravimetrically is used to calculate the percent of
drug applied to the stent in coating which is recovered following the
post curing process. Percent drug recovery data is shown in FIG. 18. This
data illustrates that 100% of the drug is recovered after spray coating
the stent and prior to post curing. However, drug recovery drops with
post curing and as post curing time increases. The data also shows that
the rate at which drug recovery drops over time is directly influenced by
the temperature at which post curing is carried out.

Example 6A

Influence of Curing Time on the Crosslink Density of a Pre-Cured Fish Oil
as Measured by Viscosity

[0205]The effect of curing time on pre-cured fish oil viscosity was
evaluated and quantified. Pre-cured fish oil (PCFO) was prepared by
heating fish oil (Ocean Nutrition 18/12TG fish oil) in a reactor at
93° C. for a total of 33 hours, while infusing oxygen through a
diffuser. During this reaction, oxidation of the oil occurs and cross
links are formed. The duration of the reaction directly influences the
extent of oxidation and cross linking that occurs, and results in an
increase in viscosity of the oil over time during the reaction. Thus the
final viscosity of the pre cured fish oil can be correlated to the extent
of cross linking and oxidation. Fish oil was reacted at 93° C.
with oxygen for 23 h, 26 h, 30.5 h, and 33 h. Viscosity was measured at
22° C. The results (FIG. 19) show an increase in viscosity
corresponds to longer reaction times and thus indirectly confirm that
increase crosslink density associated with higher viscosities also
increases with the time of curing.

Example 7

Method of Producing a Fatty Acid Based, Pre-Cure Derived Coating on a
Stent Using the Therapeutic Agent Compound C

[0206]Pre-cured fish oil (PCFO) was prepared by heating fish oil (Ocean
Nutrition 18/12TG fish oil) in a reactor at 93° C. for a total of
23 hours, while infusing oxygen through a diffuser. The resultant
viscosity of the pre-cured fish oil was 1×106 cps as measured
at 22° C. The Compound C drug coating formulation consisting of
70% Compound C, 22.5% PCFO, and 7.5% Vitamin E (DSM Nutritional Products)
was made by combining 3.7561 g PCFO and 1.2567 g Vitamin E to form a 75%
PCFO, 25% Vitamin E base coating. 15.04 g methyl-tert-butyl-ether (MTBE)
was added to produce a base coating solution of 75% solvent, 25% solids.
This solution was vortexed for 30 min until clear. Next, 767.6 mg of the
base coating solution was added to 447.8 mg Compound C. This mixture was
diluted with 7.84 g methyl-tert-butyl-ether (MTBE) to produce a final
solution of 7.6% solids for spray coating. Atrium Cinatra® CoCr stents
(3.5×13 mm) were spray coated using a Badger airbrush equipped with
a medium sized needle. The target coating load was 100 μg Compound C
per stent. Each stent was spray coated for 1.5 seconds using an air
pressure of 30 psi, while the stent was rotated. This yielded an average
coating load of 95.2 μg Compound C. Coated stents were cured in an
oven set to 93° C. for 7 hours. This process yields a conformal
stent coating having a smooth surface characteristic when imaged using
scanning electron microscopy (SEM). FIG. 20A is an SEM of the Compound C
drug coated stent at 50× magnification following 7 hours of post
curing at 93° C.

[0208]Each individual formulation component (i.e., pre-cured fish oil,
vitamin E, and Compound B) was studied before and after final curing to
understand the effects of the process on the chemistry of the Compound B
oil-derived, pre-cured biomaterial coating. In this set of experiments,
each individual component was sprayed on coupons after being diluted in
methyl-tert-butyl-ether (MBTE) to mimic coated stents. The separate
components were analyzed using appropriate spectroscopic and
chromatographic techniques before and after final curing at 200°
F. for six hours.

[0209]The effects of dissolving pre-cured fish oil into MTBE solvent and
spraying it onto a stainless steel coupon before and after final curing
using FTIR analysis is presented in FIGS. 21A, 21B, and 21C. FTIR
analysis of the pre-cured fish oil before and after curing reveals that
the double bonds in the oil are oxidizing and forming lactone/ester
cross-links resulting from the 6 hr curing process. Oxidation is
evidenced by an increase in OH band absorption and a decrease in the cis
and trans C═C peaks (FIGS. 21A and 21C), and broadening of the
carbonyl peak indicating the formation of carbonyl byproducts (FIG. 21B).
Evidence of cross-linking in the final cure is observed by the increase
in the lactone/ester peak absorption band (FIG. 21B). GC fatty acid
profile analysis of coupons is also consistent with oxidation of the oil
before and after the final curing process. FIG. 22 presents the fatty
acid compositional profile of pre-cured fish oil sprayed onto coupons
before and after the curing process. The GC fatty acid profile results
show a shift in the profile showing a reduction of unsaturated fatty
acids and an increase in the saturated fatty acids after final curing
(FIG. 22), which is consistent with oxidation of the unsaturated fatty
acids. This result is also reflected in the pre and post curing GC
chromatograms where the C16:1 and C18:1 unsaturated fatty acids peaks are
reduced in the chromatogram (FIG. 23).

[0210]FTIR spectra of vitamin E dissolved in MTBE and sprayed onto coupons
with and without final curing are presented in FIGS. 24A, 24B and 24C.
FTIR results show that the final curing process results in oxidation
which is supported by the formation of a peak at 1800-1600 cm-1
(FIG. 24C). This result is further supported by the loss of the phenol OH
absorption band after the final curing step (FIG. 24B), which occurs
after oxidation of vitamin E. The vitamin E coated onto coupons before
and after curing was extracted off the coupons and assayed by HPLC (Table
8). Each test represents an average of three samples. The results of this
study show that when vitamin E is cured (i.e., oxidized) the recovery is
reduced to 81%. HPLC chromatograms of a vitamin E control overlaid with
vitamin E sprayed onto coupons before and after curing at 292 nm are
presented in FIGS. 25A, 25B and 25C.

[0211]FTIR analysis of Compound B drug powder after spraying onto coupons
before and after curing is presented in FIGS. 26A, 26B and 26C. FTIR
reveals that dissolving Compound B in MBTE solvent and spraying it onto
the coupons changes the conformation of the drug's structure in
comparison to the control drug power spectrum. Specifically, the FTIR
results show that following the spray process the amide band is shifted
to the left and showing beginning signs of peak splitting in comparison
to the Compound B powder control (FIG. 26B). This appears to correlate
with the peak at ˜1375 cm-1 in the fingerprint region that has
changed shape in comparison to the control sample (FIG. 26c, Peak1).
There is also a peak that is formed that is not present in the control
sample at ˜1280 cm-1 (FIG. 26c, Peak2). Following the curing
of the Compound B samples several other spectral changes can be noted.
The carbonyl band merges from two peaks into one peak (FIG. 26B). This
peak is significantly broader than the Compound B powder control (FIG.
26B). Furthermore, the C--O peak at ˜1025 cm-1 disappears
following the curing process (FIG. 26c, Peak3). These changes indicate a
structural change in the Compound B as a result of curing. There is a
change in the trans C═C triene peak at about 990 cm-1 where it
is greatly reduced in intensity after curing (FIG. 26c), which indicates
that oxidation of the Compound B occurred. These changes indicate a
structural change in the Compound B structure.

[0212]Assay of the Compound B drug load by HPLC reveals an equal recovery
of the Compound B before and after spraying the Compound B onto coupons,
before final curing. Upon inspection of the native chromatogram (FIG.
27B) there does not appear to be any significant degradation products
formed after spraying. (FIG. 27A is the control.) However, following the
curing process, the Compound B drug powder recoveries is reduced to
approximately 9% (Table 9) and several new byproduct peaks are formed as
detected by HPLC (FIG. 27C). These results are consistent with the FTIR
data presented in FIG. 26c, which indicates that a degradation of the
Compound B occurred after the final curing process.

[0213]In summary, these studies showed that cured separately each
component of the oil-derived biomaterial coating was oxidized during the
curing process and specifically the Compound B therapeutic compound not
in an oil-derived stent coating formulation was significantly degraded as
a result of the curing process. This evidences the protective nature of
the pre-curing process described herein in accordance with the present
invention.

[0214]In this series of experiments the changes in chemistry for each
component in the Compound B oil-derived, pre-cured biomaterial coating
was studied. The components were mixed together, sprayed onto coupons,
and subjected to different stages of the stent coating manufacturing
process. The 60% Compound B in 30% pre-cured fish oil, and 10% vitamin E
was dissolved into MTBE and spray coated onto coupons. Samples were
analyzed before and after final curing at 93° C. for 6 hours.
FTIR, HPLC, and GC analyses were performed on the coatings. FIGS. 28A,
28B and 28C present the FTIR spectra of the Compound B oil-derived,
pre-cured biomaterial coating before and after final curing.
Interestingly, there are few spectral changes in the spectrum after final
curing, especially in functional groups assigned to the Compound B
therapeutic compound. Specifically there are no apparent intensity
changes in the trans C═C band and there does not appear to be any
significant changes in structure in the FTIR fingerprint region (FIG.
28C). The biggest difference in the Compound B biomaterial coating after
heating is an increase in absorption around 1780 cm-1, which is
consistent with lactone/ester cross-linking of the fish oil component of
the curing, and which was also observed with the fish oil by itself after
curing (FIG. 21B). These results contrast those results obtained for the
Compound B powder alone where significant structural changes were noted
after final curing (FIG. 26) and show that Compound B is more chemically
stable mixed in the oil-derived biomaterial formulation.

[0215]Further evidence for the preservation of the drug structure in the
Compound B is shown by the Compound B HPLC assay results presented in
Table 10. The average Compound B recovery in formulation is approximately
62% on coupons where it was only approximately 9% when only present as a
drug powder. Average vitamin E recovery in the coating, however, trends
lower from 81% by itself after final cure down to 68% in formulation.
Analysis of the Compound B HPLC chromatograms from the fatty-acid based,
pre-cure-derived coating formulation indicates that drug degradation is
greatly reduced in comparison to the drug only results (FIG. 27C versus
FIG. 29B).

[0216]GC fatty acid compositional analysis of the Compound B oil-derived
biomaterial formulation before and after final curing revealed that there
was a marked reduction of oxidation of the fatty acids in the fish oil in
formulation in comparison to when cured by itself (FIG. 23 versus FIG.
30). Because vitamin E is an antioxidant (i.e., oxidizes at a faster rate
than fish oil) this result is not unexpected. The inhibition of oxidation
of the unsaturated fatty acids of the oil component is also observed in
the native GC chromatogram before and after final curing (FIG. 12).

[0217]In this experiment, 60% Compound B in 30% pre-cured fish oil, and
vitamin E was dissolved into MTBE and spray coated onto 3.5×13 mm
cobalt chromium stents in order to correlate the model coupon experiments
in Examples 3 and 4 to the Compound B oil-derived biomaterial stent
coating process. The coated stents were placed in the oven to cure at
200° F. and samples were removed every hour of the 6 hour curing
process. FTIR spectroscopic analysis was performed at every time point.
Compound B and vitamin E HPLC assay testing was performed at T=0, 3, and
6 hr curing points.

[0218]FTIR analysis performed at various time points (FIGS. 31A, 31B and
31C) revealed a similar trend in final chemistry to those obtained for
the Compound B oil-derived formulation on coupons (FIG. 28).
Specifically, only small shifts in absorption bands could be noted, but
no extreme loss in Compound B structure was noted in comparison to the
Compound B drug powder FTIR coupon experiments (FIG. 26 versus FIG. 31).
HPLC assay testing performed on the stent samples at T=0, 3, and 6 hrs
for the Compound B is presented in FIG. 17. The Compound B shows
decreased recovery as a function of cure time with final curing recovery
averaging 75% for Compound B due to oxidation of the drug, but to a much
less degree than the Compound B drug powder subjected to heating alone,
which only yielded a 9% recovery. The vitamin E assay results trend
similarly to the Compound B where there is a decrease in recovery as a
function of time with a final recovery of 69% being obtained.

[0219]From the experiments performed in Examples 11-13 several conclusions
can be drawn from the data to elucidate the chemistry of formation of the
Compound B oil-derived, pre-cured biomaterial stent coating. In Example
11, each formulation component in MTBE was sprayed onto coupons and then
post cured onto the coupon surface in order to determine the changes in
chemistry for each component in the process. Analysis of the pre-cured
fish oil only coupons revealed that final curing further oxidizes the
double bonds present, as no cis C═C bonds are retained. Additional
lactone cross-linking and carbonyl byproduct formation are detected. GC
fatty acid profile analysis of the pre-cured fish oil only coupons before
and after post-curing was also consistent with oxidation of the fish oil
fatty acid double bonds. FTIR analysis of the vitamin E only coupons also
determined that the final curing process resulted in oxidation of the
vitamin E. This was evidenced by the formation of a vitamin E byproduct
peak in the carbonyl absorption region and a loss of the phenol OH
absorption band. Oxidation was confirmed by HPLC assay, which showed a
20% loss of vitamin E recovery after final curing. Finally, testing of
the Compound B drug powder sprayed onto coupons showed that that
dissolving the drug in solvent and spraying it onto the coupons is
changing the Compound B structure when compared to the control powder
spectrum (i.e., shift in amide absorption band). After final cure the
Compound B drug powder chemical structure is significantly altered as
evidenced by several absorption mode changes by FTIR, only 9% recovery of
the Compound B was obtained by HPLC assay, and byproduct peaks indicating
degradation were present in the HPLC chromatogram. Although the results
of these studies clearly show oxidation of the vitamin E, pre-cure fish
oil, and Compound B drug powder components through the final curing
process, these results suggest that that there is an additional
interaction between the formulation components (i.e., vitamin E and
pre-cured fish oil) when mixed together then when subjected to the final
curing process as typical Compound B HPLC assay results range between
75-85% recovery of the Compound B from oil-derived stent coatings.

[0220]In the second group of experiments, the Compound B, pre-cured fish
oil and vitamin E were mixed in MTBE and sprayed onto coupons, and
samples were analyzed before and after the final post-cure process step.
These data revealed a significant increase in the preservation of the
drug structure as evidenced in the FTIR spectra and the greater of
recovery of drug from the coating (i.e., ˜62%) as determined by
HPLC assay. Byproduct peaks for the Compound B were still detected by
HPLC, but were much less intense than those detected when the drug was
subjected to the final post-cure process by itself. The pre-cured fish
oil, when formulated with vitamin E, showed a decrease in oxidation in
formulation when compared to the pre-cured fish oil with no vitamin E as
detected in GC fatty acid compositional analysis (FIG. 23 vs. FIG. 12).
However, lactone/ester cross-linking was still observed.

[0221]In general, the results obtained from the stent studies mirrored
those obtained from the coupon formulation studies, except that the
average Compound B recovery was increased to 75%.

[0222]Based on the experiments conducted in this study several conclusions
can be obtained. After spraying, the coating applied to the stent appears
non-uniform and after heating the coating spreads across the surface of
the stent, the pre-cured fish oil cross-links, and a uniform coating is
produced. The recovery of the Compound B from formulation is
significantly greater than the recovery obtained when assaying the
Compound B by itself (9%), showing that the formulation (i.e., Vitamin E)
is providing some protection to the drug stability.

[0223]Analysis of the vitamin E in formulation showed that it is oxidizing
as a result of the final curing process using both FTIR and HPLC testing,
but the pre-cured fish oil is less oxidized as detected by GC fatty acid
profile. Similar to the Compound B analytical data, this result indicates
that the vitamin E is providing protection to the oil during the
oxidation process. However, despite the presence of the vitamin E,
lactone/ester cross-linking in the fish oil component in formulation on
coupons after final curing could still be detected. Lactone/ester
cross-linking can still occur in the oil-derived biomaterial coating
because the fish oil used in the formulation is pre-cured before use.
Partially curing the fish oil creates carboxyl and hydroxyl functional
groups that are needed to form lactone/ester cross-links, and thus the
presence of the vitamin E in the final curing step only serves to reduce
additional oxidation, but cannot reverse the oxidation or the molecular
species already formed in the pre-cured oil.

[0224]As can be seen, by, for example, preserving the structure of the
therapeutic agent, the therapeutic agent will have an enhanced release
profile when released from the coating.

Example 13

Method of Producing a Fatty Acid Based, Pre-Cure Derived Coating on a
Stent Using Compound E

[0225]Pre-cured fish oil (PCFO) was prepared by heating fish oil in a
reactor at 93° C. for a total of 23 hours, while infusing oxygen
through a diffuser. The resultant viscosity of the pre-cured fish oil was
1×06 cps. The coating formulation consisting of 70% Compound E,
22.5% PCFO, and 7.5% Vitamin E was made by combining 18.5 mg PCFO, 6.4 mg
Vitamin E, 57.9 mg Compound E, and 8.20 g of methyl-tert-butyl-ether
(MTBE) (Sigma Chemicals) to produce a coating solution having of 99%
solvent, 1% solids for spray coating. This solution was vortexed for 30
min until clear. Atrium Cinatra® CoCr stents (3.5×13 mm) were
spray coated using a SonoTek Medicoat DES 1000 ultrasonic Spray System.
The target coating load was 100 μg Compound E per stent with an actual
coating weight of 133.28 μg, which results in a calculated drug load
of 93.2 μg based upon the calculated final drug fraction in the
coating. Coated stents were cured in an oven set to 93° C. for 6
hours. This process yields a dry, non-tacky, conformal stent coating
having a smooth surface characteristic when imaged using scanning
electron microscopy (SEM).

[0226]In a related but separate experiment, 20 μL of the same drug
coating formulation described above was pipetted onto Cobalt Chromium
coupons with a target drug load of 100 μg of Compound E. The coated
coupons were post cured in an oven at 93° C. for 6 hours.
Gravimetric measurements of the coated coupons following the final 6 hour
post curing process demonstrated an average coating load of 152.603 μg
which based upon a calculated drug fraction of 69.92% Compound E results
in an average drug load per coupon of 106.7 μg of Compound E.
Following post curing the drug coating was extracted from the coupon in a
100% acetonitrile solution and analyzed via HPLC to determine the drug
concentration in solution, from which the total drug mass extracted from
the coupon was determined. The total drug mass extracted from the coupons
along with the actual coating weight on the coupon determined
gravimetrically is used to calculate the percent of drug applied to the
coupon in coating which is recovered following the post curing process.
The percent drug recovery from coating cured on the CoCr coupons for 6
hrs at 93° C. was calculated to be 96.7%. The drug recovery data
clearly shows that the drug integrity is preserved through the coating
formulation, application and most importantly the thermal post curing
process.

[0227]Pre-cured fish oil (PCFO) was prepared by heating fish oil in a
reactor at 93° C. for a total of 23 hours, while infusing oxygen
through a diffuser. The resultant viscosity of the pre-cured fish oil was
1×106 cps. The Compound D drug coating formulation consisting
of 50% Compound D, 37.5% PCFO, and 12.5% Vitamin E was made by combining
55.4 mg PCFO, 18.5 mg Vitamin E, 74.3 mg Compound D, and 14.67 g solvent
solution consisting of 60% methyl-tert-butyl-ether (MTBE) 40% acetone to
produce a coating solution having 99% solvent, 1% solids for spray
coating. This solution was vortexed for 30 min until clear. Atrium
Cinatra® CoCr stents (3.5×13 mm) were spray coated using a
SonoTek Medicoat DES 1000 ultrasonic Spray System. The target coating
load was 100 μg Compound D per stent with an actual coating weight of
164.16 μg, which results in a calculated drug load of 82.3 μg based
upon the calculated final drug fraction in the coating. Coated stents
were cured in an oven set to 93° C. for 6 hours. This process
yields a dry, non-tacky, conformal stent coating having a smooth surface
characteristic when imaged using scanning electron microscopy (SEM).

[0228]In a related but separate experiment, 40 μL of the same drug
coating formulation described above was pipetted onto Cobalt Chromium
coupons with a target drug load of 100 μg of sirolimus drug. The
coated coupons were post cured in an oven at 93° C. for 6 hours.
Gravimetric measurements of the coated coupons following the final 6 hour
post curing process demonstrated an average coating load of 305.59 μg
which based upon a calculated drug fraction of 50.13% sirolimus results
in an average drug load per coupon of 153.1 μg of sirolimus. Following
post curing the drug coating was extracted from the coupon in 80%
methanol, 20% (0.2% acetic acid) solution and analyzed via HPLC to
determine the drug concentration in solution, from which the total drug
mass extracted from the coupon was determined. The total drug mass
extracted from the coupons along with the actual coating weight on the
coupon determined gravimetrically is used to calculate the percent of
drug applied to the coupon in coating which is recovered following the
post curing process. The percent drug recovery from coating cured on the
CoCr coupons for 6 hrs at 93° C. was calculated to be 71.5%. The
drug recovery data clearly shows that the drug integrity is preserved
through the coating formulation, application and most importantly the
thermal post curing process. Although the sirolimus drug recovery is less
than 100% in this example, the recovery is far better than the near zero
percent recovery obtained when the pre-curing of the fish oil step is
removed and all coating curing occurs in the final coating with the drug
included.

Example 15

In-Vivo Performance and Biological Response of a Coronary Stent Coated
with a Cross Linked Fatty Acid Based Coating Incorporating a Therapeutic
Agent

[0229]In this study, a coating formulation containing 50% Compound D,
37.5% pre-cured fish oil and 12.5% tocopherol was prepared using
pre-cured fish oil having a viscosity of 1×105 cps, as
measured at 25° C. The coating formulation was subsequently
sprayed onto 3.0 mm×13 mm and 3.5 mm×13 mm Atrium Cinatra
CoCr stents using a Badger airbrush equipped with a medium sized needle.
Following the spray coating application of the coating to the stents, the
coated stents were oven post cured at 93° C. for 6 hours to
achieve a uniform and conformal drug coating layer. The final drug load
per stent, as measured by HPLC, was 68 μg of Compound D. Following
post curing, coated stents were crimped onto 3.0 mm×14 mm and 3.5
mm×14 mm PTCA catheters respectively, the devices were subsequently
packaged and sterilized via e-beam sterilization with a nominal dose of
35 kgy. The sterile drug coated stent devices were then used to conduct a
pre-clinical study in a porcine model, where by single stents were
implanted into three coronary vessels, the Left Anterior Descending
Artery (LAD), the Left Circumflex Artery (L CX) and the Right Coronary
Artery (RCA) of the porcine heart. Three groups of stents were implanted:
1) Bare metal stents, 2) stents with coating alone (no drug) and 3) drug
coated stents (DCS) to assess their comparative biological response. All
stents were implanted with an appropriate expansion to achieve a stent to
vessel diameter ratio of 1.10:1. Post implantation, animals were
recovered and were maintained for 28±2 days, at which time the animals
were sacrificed and the hearts harvested and fixed in formalin. Following
fixation, the stented vessels were isolated and dissected from the heart.
The stented arteries were dissected and embedded in methylmethacrylate
for sectioning and histopathologic evaluation. Sections were taken from
the proximal portion, mid portion, and distal portion of each stent. The
images in FIG. 32 are representative vessel cross sections having a BMS
stent. The images in FIG. 33 are representative vessel cross sections
having a stent with coating alone (no drug). The images in FIG. 34 are
representative vessel cross sections having a DCS stent. As can be seen
in these comparative images there is no notable difference in the overall
tissue reaction among the three groups with the exception of higher
levels of fibrin found in the DCS (discussed below). A comprehensive and
quantitative analysis of histomorphometry and histopathology was assessed
as part of this study, the specific results for mean injury score, mean
intimal inflammation, mean percent diameter stenosis, mean fibrin score
and % endothelialization for all three groups and are tabulated in table
11. As can be seen in table 11, the mean injury scores across the three
groups are very similar (no statistical difference), indicating that
there were no significant differences between groups in regards to the
level of mechanical vessel injury induced during stent implantation.
Generally, an injury score of less than 1 is considered to be low.
Similar to the injury scores, the mean intimal inflammation scores are
similar across all groups (no statistical difference) indicating that the
inflammation associated with both the coating alone (no drug) and the DCS
was the same as that of a stent with no coating at all. The mean %
diameter stenosis data indicates no significant difference in cellular
proliferation among groups with all groups showing low overall % diameter
stenosis at the 28 day time point. This level of cellular proliferation
among the various experimental groups is not unexpected, as the 1.10:1
overstretch results in relatively low injury during the stent
implantation process. The mean fibrin score, which is used as an
indicator for the biological drug response, clearly shows that the BMS
and coating alone groups have similarly low fibrin scores, while the
fibrin scores for the DCS group are significantly higher, indicating that
the drug has been effectively delivered to the locally stented vessel
segment, demonstrating a clear biological response to the Compound D.
This response is consistent with what has been observed with other
commercial stent products containing Compound D or analogues with a
similar mechanism of action. Lastly, the percent endothelialization
indicates the degree to which the stent and stented vessel segment is
covered with an endothelial monolayer (an endothelial monolayer being the
most intimal cell/tissue layer present in a normal functioning arterial
vessel and is critical in preventing thrombosis). The endothelialization
data shows essentially 100% re-endothelialization of the stented vessel
segment across all three groups, indicating that neither the base coating
nor drug coating interfere with the endothelial healing process.

[0230]Numerous modifications and alternative embodiments of the present
invention will be apparent to those skilled in the art in view of the
foregoing description. Accordingly, this description is to be construed
as illustrative only and is for the purpose of teaching those skilled in
the art the best mode for carrying out the present invention. Details of
the structure may vary substantially without departing from the spirit of
the invention, and exclusive use of all modifications that come within
the scope of the appended claims is reserved. It is intended that the
present invention be limited only to the extent required by the appended
claims and the applicable rules of law.

[0231]All literature and similar material cited in this application,
including, patents, patent applications, articles, books, treatises,
dissertations and web pages, regardless of the format of such literature
and similar materials, are expressly incorporated by reference in their
entirety. In the event that one or more of the incorporated literature
and similar materials differs from or contradicts this application,
including defined terms, term usage, described techniques, or the like,
this application controls.

[0232]The section headings used herein are for organizational purposes
only and are not to be construed as limiting the subject matter described
in any way.

[0233]While the present invention has been described in conjunction with
various embodiments and examples, it is not intended that the present
teachings be limited to such embodiments or examples. On the contrary,
the present invention encompasses various alternatives, modifications,
and equivalents, as will be appreciated by those of skill in the art.

[0234]The claims should not be read as limited to the described order or
elements unless stated to that effect. It should be understood that
various changes in form and detail may be made without departing from the
scope of the appended claims. Therefore, all embodiments that come within
the scope and spirit of the following claims and equivalents thereto are
claimed.